http://2009.igem.org/wiki/index.php?title=Special:Contributions/Liblint&feed=atom&limit=50&target=Liblint&year=&month=2009.igem.org - User contributions [en]2024-03-29T08:51:18ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Utah_State/PartsTeam:Utah State/Parts2009-11-20T22:11:02Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>BIOBRICKS</font></span><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<b><i>BioBricks</b></i></font><br />
<p class="class">The USU iGEM team has successfully constructed 49 BioBrick parts and devices. Of these, we have had time to demonstrate the functionality of 8 BioBricks, and had 41 of them sequenced. Check out our <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Utah_State"><font color=#009900>BioBrick Registry</font></b></a> page to see all of these parts and to find more detailed information about them. The following figures shows all of the potential different combinations of pieces used to make composite devices for this project.<br />
</p><br />
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<div align="center"> <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Utah_State"><img src="https://static.igem.org/mediawiki/2009/c/ca/Slide5USU.jpg " align = "middle" height="350" style="float:left; alt="SBC"> </a> </div><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/NotebookTeam:Utah State/Notebook2009-11-12T14:23:01Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"<font size = 4>NOTEBOOK</font></span><br />
<a href="#meeting">Meeting Notes </a><br /><br />
<a href="#notebook">Lab Notebook</a><br /><br />
<a href="#protocols">Protocols</a><br/><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Official Meetings <br />
</font></b></i> <hr><br />
<br />
<p class="header">May 12</p><br />
<br />
<p class = "class">Introduction to team members and to iGEM. Reviewed last year’s competition and last year’s team contribution. Introduction also to the 2009 iGEM home page and our team wiki. </p><br />
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<p class="header">June 23</p><br />
<br />
<p class = "class">After researching previous projects and brainstorming since our last meeting, we discussed the possibility of continuing the project that University of Hawaii initiated last year. We spoke with an advisor from their team and agreed that we could continue the project. Our team agreed that it would be a good project foundation.</p> <br />
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<p class="header">June 30</p><br />
<br />
<p class = "class">Small workshop on programming a wiki. Further collaboration with team Hawaii has taken place and we are waiting for DNA constructs with which they worked. </p><br />
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<p class="header">August 20</p><br />
<br />
<p class = "class">Discussed progress made with broad-host vector construction. The broad-host vector constructs that we have been working with from Team Hawaii and from the iGEM parts catalog do not appear to be functioning. After PCR, attempted ligations, enzymatic digestions and electrophoretic gel observations, we’ve decided to move on and try to modify another known broad-host vector to be compatible with the BioBrick format. </p><br />
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<p class="header">September 3</p><br />
<br />
<p class = "class">Team discussion of PHAs, phasin, silver-fusion, and secretory pathways. Further discussion of attempted broad-host vector modifications. </p><br />
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<p class="header">September 10</p><br />
<br />
<p class = "class">Reviewed judging criteria and reviewed our standing with the broad-host vector and the secretion pathways. Discussed which tracks would be most applicable to our project. Discussed titles for our project. Finalization of team roster and travel information. </p><br />
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<p class="header">September 17</p><br><br />
<br />
<p class = "class">Made final decisions for our intended track, chose a final project title, and gave a final review of our abstract. Discussed our wiki progress. </p><br />
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<p class="header">September 24</p><br><br />
<br />
<p class = "class">Took team picture. Presentation of different team logo options and team shirt design options. Flash animation presentation to be used potentially in wiki and presentation. <br />
<br />
<p class="header">September 29</p><br><br />
<br />
<p class = "class">Team meeting with internet programming advisor. Discussed final formatting options for wiki. </p><br />
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<p class="header">October 1</p><br><br />
<br />
<p class = "class">Instruction given on tri-parental mating. Discussed selective plates and media for tri-parental mating. </p><br />
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<p class="header">October 6</p><br><br />
<br />
<p class = "class">Instruction given on Western Blot procedure and function. Discussed modified GFP construct. Final team logo was presented.</p><br />
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<p class="header">October 8</p><br><br />
<br />
<br />
<p class = "class">Met shortly and separated to work on different assignments for wiki. </p><br />
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<p class="header">October 13</p><br><br />
<br />
<p class = "class">Final T-shirt design presented to team. Reviewed completed parts and discussed broad-host vector. Presentation of completed secretion pathway models. </p><br />
<br />
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<p class="header">October 15</p><br><br />
<br />
<br />
<p class = "class">Discussed portions of the wiki that needed to be completed before the weekend. Reviewed project components and iGEM basics in preparation for the jamboree. </p><br />
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<p class="header">October 20</p><br><br />
<br />
<br />
<p class = "class">Final meeting before wiki closure. Discussed last minute assignments to ensure that the wiki is completed.</p><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Laboratory Notebook<br />
</font></b></i> <hr><br />
<p class="class"><br />
Members of our team each had individual lab notebooks. Rather than outline each procedure that was run by each individual person, we have instead decided to present our wiki lab notebook as a weekly update of the progress that was made. These weekly updates are presented in our weekly meeting outlines, and much of the day-to-day happenings are presented in the various procedure details and specifics found below in the protocols section. </p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/1/17/Notebooksusu.jpg" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Notebooks"> </div><br />
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<b>Figure 1.</b> Some of our laboratory notebooks<br />
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Protocols<br />
</font></b></i> <hr><br />
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<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Bacterial Transformation<br />
</font></b></i> <br />
<br />
<p class="class">Once the target DNA has been successfully ligated into the plasmid vector, the plasmid must be transferred into the host cell for replication and cloning. In order to do this, the bacterial cells must first be made “competent.” The term “competent” is to describe a cell state in which there exist gaps or openings in the cell wall which will allow the plasmid containing the target genes to enter into the cell. Several methods to make bacterial cells competent exist, such as the calcium chloride method and electroporation. Competent cells may also be purchased commercially. The team at USU has purchased competent ce lls for all experiments. The following is the method used by the USU team to insert the plasmids containing various biobricks into the cells. </p><br />
<br />
<p class="header">Method</p><br> <br />
<br />
<ul class="circle"><br />
<li> Ensure the necessary antibiotic agar plates have been prepared or begin their preparation now. Four plates per transformation will be necessary (two today, then two tomorrow for streaking). Also ensure that 10 ml liquid media is made up per transformation (also for tomorrow).</li><br />
<br />
<li> If using Biobrick parts from iGEM distribution, use registry to identify appropriate will containing plasmid of interest and proceed to step 3, if using other DNA proceed to step 5.</li><br />
<br />
<li> Add 10ul of sterile water to distribution well to dissolve DNA. Remove 10ul and place in 0.5ml bullet tube. Label tube with part number, use 2ul to transform and save the other 8ul in the BioBrick part box.</li><br />
<br />
<li> Take competent cells from the -80˚C freezer and place on an ice bath.</li><br />
<br />
<li> Add 2 μl of the DNA solution (or 4ul of ligation reaction) to the competent cells. Ensure the pipetting is done directly into the cell solution. Let cells incubate on ice for 30 minutes. Heat water bath to 42˚C.</li><br />
<br />
<li> Heat shock cells in the 42˚C water bath for 30 seconds. Remove and place back in the ice bath for 2 minutes.</li><br />
<br />
<li> In the hood, add 250 μl SOC media to each tube, bringing the total cell solution to 300 μl. Incubate at 37˚C for 1 hour.</li><br />
<br />
<li> Add 200 μl of each transformed cell solution to the appropriate antibiotic plate. Use the Bunsen burner to create a “hockey stick” out of a glass pipette tip by holding over the flame until it bends. Allow to cool. Spread cell solution uniformly over the agar plate using the “hockey stick,” then before discarding, spread residual solution on the “stick” over a second plate to get more a more sparse colony distribution. </li><br />
<br />
<li> Parafilm all plates and place in 37˚C incubator 12-14 hours, or overnight if that is not possible. </li> <br />
</ul> <br />
<br />
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<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Streak Plates and Liquid Cultures from Transformed Colonies <br />
</font></b></i> <br />
<br />
<p class="class">After bacterial cells have been transformed, successfully transformed cells must be selected. Because 100% of the cells do not receive the desired plasmid and target gene, it is essential to select for cells that do have the target genes. The USU team uses antibiotic resistance to select for successful transformations. To do this, an antibiotic resistance gene is also added to the plasmid vector that contains the target genes. By doing so, it is possible to know that a cell was successfully transformed based on its ability to grow on an agar plate with antibiotics added. Because the cell is able to grow, the antibiotic resistance gene must be present as well as the target gene. From the agar plates containing the antibiotics, a colony is picked and transferred into a liquid culture for further analysis. The following is the method used by USU to clone the DNA and select for the successful transformation of various BioBricks in E.coli. </p><br />
<br />
<p class="header">Method</p><br><br />
<br />
<ul class="circle"><br />
<li> Prepare two 15 ml tubes per transformation, each with 5 ml media containing the appropriate antibiotic. </li><br />
<br />
<li> Use a pipette tip to extract half of each colony and inoculate one agar plate per colony. Using a pipette with a tip, extract the other half of each colony and inoculate one liquid media tube per colony. Label all tubes and plates and place in the 37˚C incubator until the next morning. </li><br />
</ul><br />
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<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Plasmid DNA Isolation<br />
</font></b></i> <br />
<br />
<p class="class"> Following successful bacterial cloning and isolation, it is important to verify that the target gene is in the cell and that the resultant plasmid is correct. To do this, it is a common practice to sequence the plasmid DNA. To obtain enough DNA for sequencing, the bacterial clones are grown in a liquid culture. The cells are harvested by centrifugation and then prepared for DNA plasmid extraction. DNA plasmid extraction can be done several ways, and the overall purpose is to lyse the cells and separate the plasmid DNA from all other cellular proteins, DNA, and debris. The following is the method used by the USU team to isolate plasmid DNA containing the various biobricks. </p><br />
<br />
<p class="header">Method</p><br><br />
<br />
<ul class="circle"><br />
<li> Prepare two water baths, one boiling and the other 68C.</li> <br />
<br />
<li> Centrifuge bactrerial cultures (3 to 5 ml) at 3K RPM for 20 min. Discard supernatant. </li><br />
<br />
<li> Resuspend cell pellet in 200 μl of STET buffer. Transfer to 1.5 ml tubes.</li><br />
<br />
<li> Add 10 μl of lysozyme (50 mg/ml) and incubate at room temperature for 5 min. </li><br />
<br />
<li> Boil for 45 sec and centrifuge for 20 min at 13K RPM (or until pellet gets tight).</li><br />
<br />
<li> Use a pipette tip or toothpick to remove the pellet.</li><br />
<br />
<li> Add 5 μl RNase A (10 mg/ml) to supernatant and incubate at 68C for 10 minutes.</li><br />
<br />
<li> Add 10 μl of 5% CTAB and incubate at room temperature for 3 min.</li><br />
<br />
<li> Centrifuge for 5 min at 13K RPM, discard supernatant, and resuspend in 300 μl of 1.2 M NaCl by vortexing.</li><br />
<br />
<li> Add 750 μl of ethanol and centrifuge for 5 min at 13K RPM.</li><br />
<br />
<li> Discard supernatant, rinse pellet (which cannot be seen) in 80% ethanol, and let tubes dry upside down with caps open.</li><br />
<br />
<li> Resuspend pellet in either sterile water or TE buffer. </li><br />
</ul> <br />
<br />
<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Restriction Enzyme Digestion and Electrophoresis<br />
</font></b></i> <br />
<br />
<p class="class">Restriction enzyme digestion is the process by which an insert DNA sequence is separated from the rest of the DNA molecule. Specific knowledge of the DNA insert is needed to determine which enzyme and conditions to use during the digestion reaction. Once the DNA sequence is known and the correct enzymes have been selected, the DNA may be digested. Listed below is the procedure used by USU to digest the plasmid DNA. After enzyme digestion, electrophoresis is used to separate the plasmid from the insert. A gel is prepared and the respective reaction mixes are loaded into the gel. Using a DNA ladder, and knowing the size of the insert, the corresponding band can be seen and cut out of the gel. The insert may then be removed and isolated from the gel, thus yielding the desired DNA. The DNA from this may then be used in PCR reactions, sequencing, ligations for further experimentation, etc. Listed below are example protocols used by the USU team for a restriction enzyme digestion and subsequent agarose gel electrophoresis. </p><br />
<br />
<br />
<p class="header">Method</p><br> <br />
<ul class="circle"><br />
<li> Resuspend DNA in 20 to 40 μl water, vortex, and do a brief centrifuge to get solution to the bottom of the tube. </li><br />
<br />
<li> Add components to the digestion solution in the following order: DNA (23 μl), 10X restriction enzyme buffer (3 μl), Xba1 (2 μl), and Pst1 (2 μl). The volume and restriction enzymes can be varied, but it should be ensured that the total volume is 10X the amount of RE buffer. Tap tubes periodically and allow to digest at appropriate temperature while preparing electrophoresis gel. </li><br />
<br />
<li> Prepare electrophoresis gel by adding 2 g agarose to 200 ml TAE (1% solution). This is best done in an Erlenmeyer flask of adequate volume as swirling will need to be done. Place in the microwave and microwave on high for 20 seconds at a time, pulling it out and swirling until solution is homogeneous again, then repeating (BE CAREFUL to watch the solution closely when swirling – it superheats and can boil over and cause severe burns). Continue until solution is seen boiling in the microwave then gently swirl again. </li><br />
<br />
<li> Add 20 μl ethidium bromide to solution and swirl until dissolved evenly. </li><br />
<br />
<li> Add 6 μl of 6X loading dye to each tube of digested DNA solution. </li><br />
<br />
<li> Prepare the electrophoresis unit by orienting the basin sideways with rubber gaskets firmly against the side. Place desired well template in the basin. </li><br />
<br />
<li> When the agarose solution is cool enough to comfortably touch the flask, pour into the basin until the solution is about ¾ of the way to the top of the well template. </li><br />
<br />
<li> When the gel is solidified (should look somewhat cloudy), remove the well template and change basin orientation to have the wells closest to the negative pole (as the DNA will flow towards the positive pole). Pour 1X TAE buffer into both sides of the electrophoresis unit until it just covers the gel and fills the wells.</li><br />
<br />
<li> By inserting the pipette tip below the TAE liquid and into the well, add 10 μl of DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the digested DNA solutions to the wells by adding about 2 μl less than the total volume in the tubes to prevent air bubbles in the wells.</li><br />
<br />
<li> Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 70 V (this can be as high as 100 V if time is an issue), and press the start button. Separation should take two to three hours. The yellow dye shows the location of the smaller nucleotide lengths and the blue dye shows the location of the larger nucleotide lengths. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. The basin can then be placed back in the electrophoresis unit for further separation if desired. Take care to not have the power supply on without the lid to the unit in place. </li><br />
<br />
<li> When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking. Place the cone-shaped cover over the transilluminator and place the digital camera in the top hole for pictures. </li><br />
</ul><br />
<br />
<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Media Preparation<br />
</font></b></i> <br />
<br />
<br />
<p class="class">For all experimentation involving the need for bacterial biomass and experimentation, proper media is needed to grow the cells. The following is the media composition and methods used by USU to prepare the media. </p><br />
<br />
<ul class="circle"><br />
<li> Add 5 g yeast extract, 10 g NaCl, 10 g Bacto Tryptone, and 15 g agar (if desired) to a 2 L Erlenmeyer flask and bring the volume up to 1 L with ddH20. Mix by swirling. Cover top with foil.</li><br />
<br />
<li> Autoclave for 45 minutes (liquid setting, 0 minutes drying time). It will take an additional half hour for the autoclave to finish cooling then an additional 20 to 30 minutes until the media is cool enough to pour. </li><br />
<br />
</ul><br />
<br />
<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Polymerase Chain Reaction (PCR)<br />
</font></b></i> <br />
<br />
<p class="class">PCR is used to amplify a desired DNA sequence. The reaction is first set up by designing primers that will bind only to the desired regions of the DNA sequence. Once the primer and polymerase have been selected, the reaction parameters of time and temperature must be optimized. When the reaction works properly only the target DNA will be amplified into large quantities that may then be isolated and used for further experimentation. The following is the procedure used by USU for PCR reactions to amplify various biological parts. A useful set of primers are the universal BioBrick primers VF2 and VR that can be used to amplify almost any BioBrick part. </p><br />
<br />
<p class="header">Method</p><br><br />
<ul class="circle"><br />
<br />
<li>Obtain the following reagents from the freezer: DNA template (cells or DNA), 10X Taq buffer (+KCl, -Mg/Cl2), MgCl2, 10 mM dNTP Mix, Taq polymerase (take out of freezer only immediately when needed and put back), and sterile distilled H2O. Place all reagents on ice. Also obtain PCR (either 0.2 or 0.5 ml) tubes.</li><br />
<br />
<li>Add the following reagents to a tube (50 μl reaction) in the following volumes and order:<br />
<ul type="bullet type"><br />
<li>32 μl sterile H2O,<br />
<br />
<li>5 μl 10X buffer,<br />
<br />
<li>2 μl dNTP Mix,<br />
<br />
<li>6 μl MgCl2<br />
<br />
<li>3 μl cells/DNA,<br />
<br />
<li>0.25 μl Taq Polymerase<br />
<br />
<li>1 μl primer 1<br />
<br />
<li>1 μl primer 2 <br />
</ul><br />
<br />
MgCl2 volume can be varied (lower to increase specificity – just ensure total volume is 50 ul with H2O). If many reactions are to be constructed, a master mix can be made up to cut down on time and pipette tip usage (if this is done, ensure primers are added to the appropriate reaction, i.e. perhaps not to the master mix). Tap or vortex tubes and take to the thermocyler. Place all reagents back in the -20˚C freezer.</li><br />
<br />
<li>Choose thermocycler temperatures. The Eppendorf Mastercycler will cycle between three temperatures: typical temperatures are 94˚C for denaturing, 50-60˚C for primer annealing, and 72˚C for polymerase extending. Lowering the annealing temperature decreases DNA specificity; 55˚C is a good temperature to begin if no trials have been made with the sample.</li><br />
<br />
<li>Turn on thermocycler with the switch in the back of the unit and open the lid. The placement of the tubes depends on the size of the tube (0.2 or 0.5 ml) and whether or not a temperature gradient is to be used.</li><br />
<ul type="bullet type"><br />
<li> If no temperature gradient will be used, tubes can be placed anywhere on the unit in the appropriately-sized hole. Select “Files” and press enter. Select “Load” and then “Standard.” If cells will be used in the reaction, include a 1-minute lysing step at the beginning (step 1); this will be followed by a 1-minute DNA denaturing step (step 2). If purified DNA will be used, set step 1 to 1 second. Set an annealing temperature for step 3. Ensure the lid temperature is 105˚C and the extending temperature is 72˚C. Press exit. If prompted to save, save by pressing enter three times. Press exit to return to the main menu. Choose “Start” on the main menu and select “Standard.” The program should begin.<br />
<br />
<li> If a temperature gradient is to be used, temperature will vary according to column. A 20˚C range is the maximum range that can be used (+/- 10˚C). The range is made by setting a temperature for the middle column and then setting a +/- range. To see what the temperatures will be if a gradient is used, select “OPTIONS” on the main menu, then select “Gradient.” Select the size tube that is being used by pressing “Sel,” then press enter. Choose a temperature for the center column, press enter, then select a +/- range and press enter. The column number along with the corresponding temperature is shown. Decide tube placement based on this information. Press exit twice to return on the main menu. Select “Files” then “Load,” then “Gradient.” If cells are being used, set the cell lysing step (step 1) to 1 minute (1:00); if purified DNA is being used, set this time to 1 second (0:01). Step 2 should be 94C, Step 4 should be 72˚C, and the lid temperature should be 105˚C. Go to step 3 and set an annealing temperature for the center column. Leave the next two lines as they are, and change the gradient setting (“G”) to the +/- the center temperature amount. Press exit. If prompted to save, press enter three times; if not prompted to save, press enter once. Press exit to get back to the main menu. To begin cycle, select “Start,” then select “Gradient.” The program should begin. <br />
</ul><br />
<li>The thermocycler is set to store the completed reaction tubes at 4˚C when finished. </li> <br />
</ul><br />
<br />
<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Ligation<br />
</font></b></i> <br />
<br />
<p class="class">Ligation is the process by which the insert (target DNA gene) is inserted into a plasmid. Both the plasmid and insert have been digested and have the proper “sticky” or blunt ends which are compatible for joining the two DNA pieces together into one molecule. These two DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid. This newly formed plasmid may then be isolated using gel electrophoresis and then used for bacterial transformation or other experimentation. The following is the procedure used by USU to ligate together various biobrick parts. </p><br />
<br />
<p class="header">Method</p><br> <br />
<ul class="circle"><br />
<li> 1. Obtain the following reagents, some of which are in the -20˚C freezer: DNA vector, DNA insert, 10X ligation buffer, T4 DNA ligase (take out only when needed, then return immediately to freezer), and sterile distilled water.</li><br />
<br />
<li> 2. Ideally, it is desirable to have the concentration of insert ends (or moles of insert) be two to three times the concentration of vector ends (or moles of vector), with a total DNA concentration of 50-400 ng/μl in the reaction. If determining the DNA concentration is not possible, place two to three times the volume of vector as the volume of insert in the reaction. As this is often the case, place the following reagents in a thin-walled PCR tube in the following volumes:<br />
<br />
• 10 μl insert DNA<br />
<br />
• 3 μl vector DNA<br />
<br />
• 2 μl 10X ligation buffer<br />
<br />
• 4 μl H2O<br />
<br />
• 1 μl T4 DNA ligase<br />
<br />
= 20 μl total<br />
<br />
This could also be done in different volumes depending on DNA concentration/total volume desired.</li><br />
<br />
<li> 3. Gently mix the tube, and place the tube in the PCR thermocyler, turn on the machine, select “Start,” from the main menu, select “22” and press “Start.” The thermocycler will keep the reaction at 22˚C.</li><br />
<br />
<li> 4. Incubate for 60 minutes. Heat-inactivate by placing tubes in 68C water bath for 10 minutes. Place in the freezer if storing for later use. </li><br />
<br />
</ul> <br />
<br />
<br />
<br> <br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Western Blot<br />
</font></b></i> <br />
<br />
<p class="class">Western blotting is a procedure that allows for the identification of proteins using a specific antibody after protein separation on an SDS polyacrylamide gel. </p><br />
<br />
<p class="header">Method</p><br> <br />
<br />
<ul class="circle"><br />
<li>Collect bacterial cells by centrifugation and lyse the cells using any of a variety of procedures.</li><br />
<li>Spin at 14,000 rpm (16,000 g) in a microfuge for 10 min at 4°C.</li><br />
<li>Transfer the supernatant to a new tube and discard the pellet.</li><br />
<li>Determine the protein concentration (Bradford assay, A280, or BCA)</li><br />
<li>Mix 20 µl of sample (20ug) with 10ul of 3x sample buffer.</li><br />
<li>Boil for 1 min, ool at RT for 5 min.</li><br />
<li>Flash spin to bring down condensation prior to loading gel.</li><br />
<li>Assemble pre-prepared polyacrylamide gel into gel running rig.</li><br />
<li>Load protein samples into individual wells.</li><br />
<li>Use 10 µl of Kaleidoscope standard.</li><br />
<li>Run at 35 mA (constant current) for approximately 2hrs.</li><br />
<li>Disassemble gel when done running.</li><br />
<li>Cut a piece of PVDF membrane (Millipore Immobion-P #IPVH 000 10).</li><br />
<li>Wet for about 10 min in methanol on a rocker at room temp.</li><br />
<li>Remove methanol and add 1x Blotting buffer until ready to use.</li><br />
<li>Assemble "sandwich" for Bio-Rad's Transblot.</li><br />
<li>Sponge - filter paper - gel - membrane - filter paper - sponge</li><br />
<li>Transfer for 1 hr at 100V at 4°C on a stir plate. Bigger proteins might take longer to transfer.</li><br />
<li>When finished, immerse membrane in blocking buffer (5% nonfat dry milk)and block overnight.</li><br />
<li>Incubate with primary antibody diluted in 0.5% blocking buffer for 60 min at room temp.</li><br />
<li>Wash 3 x 10 min with 0.05% Tween 20 in PBS.</li><br />
<li> Incubate with secondary antibody diluted in 0.5% blocking buffer for 45 min at room temp.</li><br />
<li>Wash 3 x 10 min with 0.05% Tween 20 in PBS.</li><br />
<li>Detect with TMB stabilized substrate for HRP. </li><br />
<br />
</ul><br />
<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Site-Directed Mutagenesis<br />
</font></b></i> <br><br />
<br />
<br />
<p class="class">QuikChange II Site-Directed Mutagenesis Kit (Stratagene) </p><br />
<br />
<ul class="circle"><br />
<li>Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence.</li><br />
<br />
<li>Prepare the control reaction as indicated below:<br />
<ul class="circle"><br />
<li>5 μl of 10× reaction buffer (see Preparation of Media and Reagents)<br />
<br />
<li>2 μl (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/μl)<br />
<br />
<li>1.25 μl (125 ng) of oligonucleotide control primer #1 [34-mer (100 ng/μl)]<br />
<br />
<li>1.25 μl (125 ng) of oligonucleotide control primer #2 [34-mer (100 ng/μl)]<br />
<br />
<li>1 μl of dNTP mix<br />
<br />
<li>39.5 μl of double-distilled water (ddH2O) to a final volume of 50 μl<br />
</ul><br />
Then add<br />
<ul class="circle"><br />
<li> 1 μl of PfuTurbo DNA polymerase (2.5 U/μl)</li><br />
</ul><br />
<li>Prepare the sample reaction(s) as indicated below: <br />
<br />
<p class="class">Note: Set up a series of sample reactions using various concentrations of dsDNA template ranging from 5 to 50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA template) while keeping the primer concentration constant. </p><br />
<ul class="circle"><br />
<li>5 μl of 10× reaction buffer<br />
<br />
<li>X μl (5–50 ng) of dsDNA template<br />
<br />
<li>X μl (125 ng) of oligonucleotide primer #1<br />
<br />
<li>X μl (125 ng) of oligonucleotide primer #2<br />
<br />
<li>1 μl of dNTP mix<br />
<br />
<li>ddH2O to a final volume of 50 μl<br />
<br />
<p class="class">Then add</p><br />
<br />
<li>1 μl of PfuTurbo DNA polymerase (2.5 U/μl) </li><br />
</ul><br />
<li>If the thermal cycler to be used does not have a hot-top assembly, overlay each reaction with ~30 μl of mineral oil.</li><br />
<br />
<li>Cycle each reaction using the cycling parameters as outlined in Table I of the Stratagene QuikChange II Site-Directed Mutagenesis Kit manual. We used an annealing temperature of 55C for 1 min and an extension temperature of 68C for 5 min and 18 cycles.</li><br />
<br />
<li>Following temperature cycling, place the reaction on ice for 2 minutes to cool the reaction to ≤37°C. If desired, amplification may be checked by electrophoresis of 10 μl of the product on a 1% agarose gel. A band may or may not be visualized at this stage. In either case proceed with Dpn I digestion and transformation. </li><br />
</ul><br />
<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Dpn I Digestion of the Amplification Products<br />
</font></b></i><br />
<ul class="circle"><br />
<li>Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction below the mineral oil overlay using a small, pointed pipet tip.</li)<br />
<br />
<li>Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37°C for 1 hour to digest the parental (i.e., the nonmutated) supercoiled dsDNA. </li><br />
<br />
Transform into XL1-Blue Supercompetent Cells and proceed as previously described. </p><br />
<br />
<br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br /><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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References<br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
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Experimental Section: Approach for BioBrick Compatibility<br />
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Converting Broad-Host Vectors into a BioBrick-Compatible Format<br />
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<p class="class">Two Broad-host range vectors were used in this study; pRL1383a and PCPP33. To convert these vectors into BioBrick-compatible format, the four standard BioBrick sites EcoRI, XbaI, SpeI, and PstI needed to be inserted into the multiple cloning site. For pRL1383a, common BioBrick primers VR and VF2 were also included to allow the use of PCR in amplifying the BioBrick parts.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/8/82/PRL1383A_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 1.</b> Plasmid map of pRL1383a <br />
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<b>Figure 2.</b> Plasmid map of pCPP33 <br />
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<p class="class">Apart from being shown effective in the Synechosystis PCC 6803 (Marraccini 1993), pRL1383a is an ideal candidate for use as a BioBrick-compatible broad-host range vector because the BioBrick restriction sites are absent within the vector sequence. To convert pRL1383a into a BioBrick format, the existing multiple cloning site, which is flanked by a BamHI site and a HindIII site, was utilized. First, modified primers were synthesized from BioBrick primers VR and VF2. These primers were modified by adding extra nucleotides to insert the desired restriction enzyme sites into the PCR product. A BamHI site was added to 5’ end of the forward primer (VF2) and a HindIII site was added to the 5’ end of the reverse primer (VR). These primers were used to amplify an existing, tested BioBrick part by PCR. For this purpose, we selected BBa_I20260 because it does not contain BamHI or HindIII sites, and successful ligation is readily testable as it is a GFP -producing construct. The addition of IPTG is typically necessary to induce GFP production in this particular device. However, when using Top10 <i>E. coli</i> cells it is produced continuously because these cells lack a lac repressor (insert invitrogen link). After cutting the vector at the multiple cloning site using BamHI and HindIII, the BioBrick insert obtained by PCR with modified ends was ligated into the backbone. The vector was then transformed using Top10 One Shot® chemically competent <i>E. coli</i> and tested for successful insertion using PCR and restriction digests.</p><br />
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<p class="class">Another broad host range vector, pCPP33, previously shown effective in Pseudomonas Putida,was standardized using similar methods. While the complete sequence of this plasmid is not available, it was shown that there are no BioBrick restriction sites outside the multiple cloning site (Figure 2). The multiple cloning site of this vector is flanked by EcoRI and HindIII. This allowed the PCR product of BBa_I20260 to again be used by cutting with HindIII and EcoRI restriction enzymes. Restriction digests and gel analysis were used to test for the insert.</p><br />
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Broad Host Conjugation<br />
</font></b></i><br />
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<p class="class">In order to transfer a vector of interest using conjugation, the <i>tra</i> gene (contained in what we will refer to as a transfer plasmid, or helper plasmid) must be expressed in order to initiate the conjugation process. This plasmid codes for genes which, when expressed, form pili on the cell surface, which in turn initiate conjugation (Heinemann 1989). This plasmid may be present in one of three different procedures:</p><br />
<ul><br />
</li><li><b>Hfr strain</b> – The <i>tra</i> operon is many times contained in an episome, which can incorporate itself into the cell genome. These resultant Hfr strains will often begin the transfer of their own DNA, both plasmid and genomic. Due to the transfer of the genomic DNA, these strains are referred to as high frequency recombinant (Hfr) strains.<br />
<br />
</li><li><b>Biparental (normal) Conjugation</b> – Cells containing the <i>tra</i> genes, often labeled as F-positive (F+) due to the F-plasmid, a well-known transfer plasmid, can express the transfer genes necessary for conjugation to occur. When a vector of interest and a transfer plasmid are of different incompatibility groups, they may both be transformed into the same cell, and conjugation may take place between the F+ donor cell and the recipient cell<br />
<br />
</li><li><b>Triparental Mating</b> – In the case where the transfer plasmid and the vector of interest are of the same incompatibility group, the two plasmids may not stably coexist (Heinemann 1989). In this case, two separate cells containing the transfer gene (the helper cell) and the vector (the donor cell) must be used in conjugation. The helper cell will assist the donor cell in the transfer of its mobilizable plasmid to the recipient cell. This method circumvents some of the barriers that may prevent the transfer of plasmids.<br />
</ul><br />
<br />
<br />
<p class="class">For our project, we chose to use the triparental mating procedure for the transmission of our vector. While not being the most efficient method, it circumvents possible barriers and intermediate steps.</p><br />
<br />
<p class="class">Because of the use of three different cells in our transformation procedure, the selection criteria for each component needed to be unique. In addition, we selected helper plasmids which had been known to work with the intended recipient cell.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/4a/PCPP33_tri-p_table.png" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Table 1</b> Components and selection criteria used in conjugation with the broad-host vector PCPP33 <br />
</div><br />
<br><br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f2/PRL1383A_tri-p_table.png" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Table 2</b> Components and selection criteria used in conjugation with the broad-host vector PRL1383A <br />
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<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Results<br />
</font></b></i><br />
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<br />
<p class="class">Testing the ligation of pRL1383a and BBa_I20260 using PCR and restriction digests showed that the insert was not present in the vector, and the conversion to BioBrick format ultimately unsuccessful. The procedure as described above was repeated multiple times without success. Tri-parental conjugation of unmodified pRL 1383a was inconclusive in all target organisms.</p><br />
<br />
<p class="class">In an effort to troubleshoot this vector, several different approaches were taken. First, the ligation was repeated with varying concentrations of insert (10X, 2X) in an attempt to account for the impact of the large vector size on the ligation reaction. These ligations yielded similar results to reactions done at calculated concentrations. A Blunt-end ligation using a Klenow fragment was also performed. This was repeated, both attempts without success. The BBa_I20260 PCR product with BamHI/HindIII ends was ligated into another vector in an attempt to test the insert’s ability to be cut with the restriction enzymes. This ligation did not indicate the presence of the insert, suggesting that the problem lies with the vector or primers. The primers were tested and found viable on another insert, with similar testing of restriction enzymes to show functionality. The primers and enzymes were operating as intended, but new enzymes were ordered for more experimental certainty. The insert was then digested only with HindIII, and left in a ligation reaction. The outcome of this ligation was not of the desired length. This was repeated, and the same result obtained. While there is some suggestion that the BioBrick insert may not be functioning, the ambiguous results of tri-parental mating with unmodified pRL1383a suggests that the vector may be damaged or misunderstood.</p><br />
<br />
<p class="class">Testing the ligation of PCPP33 and BBa_I20260 also proved unsuccessful. Restriction digests using BioBrick standard pieces failed to yield an insert. Tri-parental mating of this vector proved successful in all organisms that we tested. All organisms yielded colonies on tetracycline plates, suggesting presence of the plasmid. Further testing by plasmid extraction and gel analysis will be done to conclusively determine presence of the plasmid. <br />
</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/7/77/R_spaeroides_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/1/1e/P_putida_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/d/da/Synechocystis_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 3.</b> Results of the tri-parental mating between pCPP33 and R. <i>sphaeroides</I>, P. <i>putida</i>, and Synechocystis sp., respectively. Each plate is shown alongside a negative control <br />
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Experiments: Secretion<br />
</font><br></b></i><hr><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20><br />
Methods for Constructing BioBrick Parts<br />
</font><br></b><br />
<br />
<p class="class"><br />
One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length. <br />
<br />
<p class="class"><br />
Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.</p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Signal Peptides:</font></b><br><br />
<p class="class"><br />
To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.</p><br />
<p class="class"><br />
The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Phasin:</font></b><br><br />
<p class="class"><br />
The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis. Sequencing confirmed that this site was successfully removed. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>GFP:</font></b><br><br />
<p class="class"><br />
Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported. Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator. </p><br />
<br />
<p class="class"><br />
A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure 4 shows GFP- Top10 <i>E. coli</i> colonies (left) and unfused GFP+ Top10 <i>E. coli</i> colonies (right). This figure shows that the GFP construct is functional and easily detectable.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/93/GFPglowingUSU.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 4.</b> Plate with GFP- cells (left) next to plate with GFP+ cells(right)<br />
</div><br />
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<br />
<br><b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Bioplastic Production:</font></b><br><br />
<p class="class">A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. <i>E. coli</i> harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure 5. The observed peaks at 1.24 ppm, 2.54 ppm, and 5.2 ppm correspond with those observed in standard polyhydroxyalkanaote samples.</p><br />
<Br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/4/43/NMRusu.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 5.</b> Proton NMR spectra for PHB production in recombinant <i>E. coli</i><br />
</div><br />
<br><br />
<p class="class">To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>SDS-PAGE Analysis</font></b><br><br />
<p class="class">Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged (see Figure 6). Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure 6. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/d/d1/GFP_gel.png"" align = "middle" height="400" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 6.</b> Protein gel showing a strong band corresponding to GFP<br />
</div><br />
<br><br />
<p class="class">The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection, as shown in Figure 7. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/3/3e/PHB_gel.png"" align = "middle" height="250" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 7.</b> Protein gel showing the presence of phasin protein in supernatant samples (third well from left)<br> next to supernatant from an <i>E. coli</i> sample without a phasin-producing construct.<br />
</div><br />
<Br><br />
<br />
<p class="class">Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies. </p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/SecretionTeam:Utah State/Secretion2009-11-12T04:28:43Z<p>Liblint: </p>
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant <i>E. coli</i> MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing <i>E. coli</i> transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing <i>E. coli</i> or photoautotrophic PHA-producers <i>R. sphaeroides</i> and <i>Synechocystis</i> PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure 1 depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 1.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant <i>E. coli</i> do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
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<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure 2 depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as <i>E. coli</i> K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure 3. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative <i>E. coli</i> (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure 3 illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 3.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure 4 shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 4.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of <i>E. coli</i> (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
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<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in <i>E. coli</i> harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
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<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in <i>E. coli</i> (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 5 depicts the GFP barrel structure.</p><br />
<br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="200" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 5.</b> The GFP Barrel Structure<br />
</div><br />
<br />
<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In <i>E. coli</i>, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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<table width="100%" border="0"><br />
<tr><br />
<td width="16%" valign="top"><table width="72%" border="0"><br />
<tr valign="top"><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>JUDGING</font></span><br />
<a href="#bronze">Bronze</a><br /><br />
<a href="#silver">Silver</a><br /><br />
<a href="#gold">Gold</a><br /><br />
</td> <br />
</tr><br />
<br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
</tr><br />
</table></td><br />
<td><table width=100% style="background:#CCCCCC; padding:7px; border-style:none"><br />
<tr><br />
<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>JUDGING CRITERIA</b></i></font><br />
<HR><br />
<br />
<font size="3" face="Helvetica, Arial, San Serif" color =#231f20><b><i>In fulfillment of the requirements for the Gold Medal, the 2009 Utah State iGEM Team did the following:</b></i></font><br><br />
<br><br />
<a name="bronze"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Bronze Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Completed the registration requirements and Project Summary form.</li><br />
<li>Prepared and will present a poster and talk at the 2009 Jamboree.</li><br />
<li>Entered all necessary information detailing 62 BioBricks into the Registry of Standard Parts.</li><br />
<li>Designed parts in conformity with accepted BioBrick standards.</li><br />
<li>DNA for 49 BioBricks entered in the Registry were sent in to iGEM headquarters. The majority of these parts were confirmed with DNA sequencing.</li><br />
</ul><br />
<br><br />
<br />
<a name="silver"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Silver Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Demonstrated that several submitted BioBricks work as expected.</li><br />
<p class="margin">- The composite lac promoter/RBS/GFP/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208045"><b><font color=#009900>BBa_K208045</font></b></a>). This composite was demonstrated by the presence of GFP visualized on a UV transilluminator. The GFP protein was also visualized using SDS polyacrylamide gel electrophoresis. See pictures on Wiki. </p> <br />
<p class="margin">- New GFP reporter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208000"><b><font color=#009900>BBa_K208000</font></b></a>). This reporter, with an excitation/emission of 395/509, was shown to be functional using the novel composite construct BBa_K2208045 (lac promoter/RBS/GFP/terminator). Picture on Wiki under part BBa_K2208045. </p><br />
<p class="margin">- The composite lac promoter/RBS (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010"><b><font color=#009900>BBa_K208010</font></b></a>). This was demonstrated using the composite construct BBa_K2208045. Picture on Wiki under part BBa_K2208045. This composite part is extremely useful because it alleviates the need to work with extremely small ribosomal BioBrick components. </p> <br />
<p class="margin">- Lac promoter/RBS/Gene III/Phasin/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208038"><b><font color=#009900>BBa_K208038</font></b></a>). This was demonstrated through the detection of phasin proteins isolated from supernatant samples using SDS polyacrylamide gel electrophoresis. Picture on Wiki. </p><br />
<p class="margin">- Phasin gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208001"><b><font color=#009900>BBa_K208001</font></b></a>). A protein of the correct size was detected in SDS polyacrylamide gel electrophoresis from cells having this construct that was not detected in the control samples. <br />
<p class="margin">- Gene III secretion tag (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208002"><b><font color=#009900>BBa_K208002</font></b></a>). The phasin protein from part BBa_K208038 was detected outside the cell, thus demonstrating the functionality of this Gene III secretion tag.</p><br />
<li>Characterized the GFP reporter (BBa_K208000) (1) showing GFP expression over time and (2) showing sensitivity of IPTG induction.</li><br />
</ul><br><br />
<br />
<a name="gold"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Gold Medal Requirements</b></font><br />
<Br><br />
<ul class="circle"><br />
<li>Improved upon the existing set of GFP BioBrick parts by constructing a GFP with a larger Stokes shift (395/509nm), making for easier downsteam analysis. The bright fluorescence can very easily be seen on a standard UV transilluminator. As GFP is one of the most commonly used reporters, the introduction of this new and improved BioBrick part should be of great benefit to the iGEM community.</li> <br />
<li>Our initial plans for our 2009 team was in part to continue the 2008 Hawaii team’s project, “Cyanobacteria Toolkit,” by making the pRL1383a vector an effective broad-host vector. When this vector proved ineffective, we made efforts to troubleshoot. We had much correspondence with the advisors of the Hawaii team, including a conference call mid-summer. Since most of their parts were not included in the 2009 iGEM distribution, we had them send many of the parts they used. After numerous approaches and attempts to convert pRL1383a into BioBrick format, we finally decided that the vector was either mischaracterized or altered in some unknown way. </li> <br />
<li>As thoroughly explained in our Wiki, one of the primary goals of our project has been to expand the BioBrick world to organisms other than E. coli. We have documented the many benefits that would come from such an achievement. Though the pRL1383a vector efforts were unsuccessful, we did successfully express the vector pCPP33 in E. coli, Pseudomonas putida, Synechocystis pcc 6803, and Rhodobacter sphaeroides. This vector now has only to be put in BioBrick format. </li> <br />
<li>In reference to our ethics section (https://2009.igem.org/Team:Utah_State/ETHICS), our team has taken specific measures to follow our suggested proposals. In the education of our team, we discussed the potential benefits of a standard secretion system but also discussed the potential of our designed secretion pathways to be used in a malevolent manner. As a team, we acknowledge the importance of high moral accountability and commitment to safety and security. Additionally, in an effort to foster the sharing of information in our community, upon completion of the jamboree we will submit an article to be released in our school newspaper and in the College of Engineering website. This winter we are also hosting a lecture by Drew Endy addressing synthetic biology which will be open to the public. We hope to act as ambassadors to foster support and excitement in our own community.</li> <br />
<br />
</td><br />
</tr><br />
</table><br />
</tr><br />
</table><br />
</body><br />
</html><br />
<br />
<html><br />
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<style><br />
table {<br />
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</html></div>Liblinthttp://2009.igem.org/Team:Utah_State/AchievementsTeam:Utah State/Achievements2009-11-12T04:20:10Z<p>Liblint: </p>
<hr />
<div><html><br />
<a href="https://2009.igem.org/Team:Utah_State"><br />
<img alt="USU iGem" src="https://static.igem.org/mediawiki/2009/7/71/USUlogo.jpg"/> </a><br />
<br />
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<table width="100%" border="0"><br />
<tr><br />
<td width="16%" valign="top"><table width="72%" border="0"><br />
<tr valign="top"><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>JUDGING</font></span><br />
<a href="#bronze">Bronze</a><br /><br />
<a href="#silver">Silver</a><br /><br />
<a href="#gold">Gold</a><br /><br />
</td> <br />
</tr><br />
<br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
</tr><br />
</table></td><br />
<td><table width=100% style="background:#CCCCCC; padding:7px; border-style:none"><br />
<tr><br />
<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>JUDGING CRITERIA</b></i></font><br />
<HR><br />
<br />
<font size="3" face="Helvetica, Arial, San Serif" color =#231f20><b><i>In fulfillment of the requirements for the Gold Medal, the 2009 Utah State iGEM Team did the following:</b></i></font><br><br />
<br><br />
<a name="bronze"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Bronze Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Completed the registration requirements and Project Summary form.</li><br />
<li>Prepared and will present a poster and talk at the 2009 Jamboree.</li><br />
<li>Entered all necessary information detailing 62 BioBricks into the Registry of Standard Parts.</li><br />
<li>Designed parts in conformity with accepted BioBrick standards.</li><br />
<li>DNA for 49 BioBricks entered in the Registry were sent in to iGEM headquarters. The majority of these parts were confirmed with DNA sequencing.</li><br />
</ul><br />
<br><br />
<br />
<a name="silver"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Silver Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Demonstrated that several submitted BioBricks work as expected.</li><br />
<p class="margin">- The composite lac promoter/RBS/GFP/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208045"><b><font color=#009900>BBa_K208045</font></b></a>). This composite was demonstrated by the presence of GFP visualized on a UV transilluminator. The GFP protein was also visualized using SDS polyacrylamide gel electrophoresis. See pictures on Wiki. </p> <br />
<p class="margin">- New GFP reporter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208000"><b><font color=#009900>BBa_K208000</font></b></a>). This reporter, with an excitation/emission of 395/509, was shown to be functional using the novel composite construct BBa_K2208045 (lac promoter/RBS/GFP/terminator). Picture on Wiki under part BBa_K2208045. </p><br />
<p class="margin">- The composite lac promoter/RBS (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010"><b><font color=#009900>BBa_K208010</font></b></a>). This was demonstrated using the composite construct BBa_K2208045. Picture on Wiki under part BBa_K2208045. This composite part is extremely useful because it alleviates the need to work with extremely small ribosomal BioBrick components. </p> <br />
<p class="margin">- Lac promoter/RBS/Gene III/Phasin/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208038"><b><font color=#009900>BBa_K208038</font></b></a>). This was demonstrated through the detection of phasin proteins isolated from supernatant samples using SDS polyacrylamide gel electrophoresis. Picture on Wiki. </p><br />
<p class="margin">- Phasin gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208001"><b><font color=#009900>BBa_K208001</font></b></a>). A protein of the correct size was detected in SDS polyacrylamide gel electrophoresis from cells having this construct that was not detected in the control samples. <br />
<p class="margin">- Gene III secretion tag (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208002"><b><font color=#009900>BBa_K208002</font></b></a>). The phasin protein from part BBa_K208038 was detected outside the cell, thus demonstrating the functionality of this Gene III secretion tag.</p><br />
<li>Characterized the GFP reporter (BBa_K208000) (1) showing GFP expression over time and (2) showing sensitivity of IPTG induction.</li><br />
</ul><br><br />
<br />
<a name="gold"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Gold Medal Requirements</b></font><br />
<Br><br />
<ul class="circle"><br />
<li>Improved upon the existing set of GFP BioBrick parts by constructing a GFP with a larger Stokes shift (395/509nm), making for easier downsteam analysis. The bright fluorescence can very easily be seen on a standard UV transilluminator. As GFP is one of the most commonly used reporters, the introduction of this new and improved BioBrick part should be of great benefit to the iGEM community.</li> <br />
<li>Our initial plans for our 2009 team was in part to continue the 2008 Hawaii team’s project, “Cyanobacteria Toolkit,” by making the pRL1383a vector an effective broad-host vector. When this vector proved ineffective, we made efforts to troubleshoot. We had much correspondence with the advisors of the Hawaii team, including a conference call mid-summer. Since most of their parts were not included in the 2009 iGEM distribution, we had them send many of the parts they used. After numerous approaches and attempts to convert pRL1383a into BioBrick format, we finally decided that the vector was either mischaracterized or altered in some unknown way. </li> <br />
<li>As thoroughly explained in our Wiki, one of the primary goals of our project has been to expand the BioBrick world to organisms other than E. coli. We have documented the many benefits that would come from such an achievement. Though the pRL1383a vector efforts were unsuccessful, we did successfully express the vector pCPP33 in E. coli, Pseudomonas putida, Synechocystis pcc 6803, and Rhodobacter sphaeroides. This vector now has only to be put in BioBrick format. </li> <br />
<li>In reference to our ethics section (https://2009.igem.org/Team:Utah_State/ETHICS), our team has taken specific measures to follow our suggested proposals. In the education of our team, we discussed the potential benefits of a standard secretion system but also discussed the potential of our designed secretion pathways to be used in a malevolent manner. As a team, we acknowledge the importance of high moral accountability and commitment to safety and security. Additionally, in an effort to foster the sharing of information in our community, upon completion of the jamboree we will submit an article to be released in our school newspaper and in the College of Engineering website. This winter we are also hosting a lecture by Drew Endy addressing synthetic biology which will be open to the public. We hope to act as ambassadors to foster support and excitement in our own community.</li> <br />
<br />
</td><br />
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<img alt="USU iGem" src="https://static.igem.org/mediawiki/2009/7/71/USUlogo.jpg"/> </a><br />
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<table width="100%" border="0"><br />
<tr><br />
<td width="16%" valign="top"><table width="72%" border="0"><br />
<tr valign="top"><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>JUDGING</font></span><br />
<a href="#bronze">Bronze</a><br /><br />
<a href="#silver">Silver</a><br /><br />
<a href="#gold">Gold</a><br /><br />
</td> <br />
</tr><br />
<br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
</tr><br />
</table></td><br />
<td><table width=100% style="background:#CCCCCC; padding:7px; border-style:none"><br />
<tr><br />
<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>JUDGING CRITERIA</b></i></font><br />
<HR><br />
<br />
<font size="3" face="Helvetica, Arial, San Serif" color =#231f20><b><i>In fulfillment of the requirements for the Gold Medal, the 2009 Utah State iGEM Team did the following:</b></i></font><br><br />
<br><br />
<a name="bronze"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Bronze Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Completed the registration requirements and Project Summary form.</li><br />
<li>Prepared and will present a poster and talk at the 2009 Jamboree.</li><br />
<li>Entered all necessary information detailing 62 BioBricks into the Registry of Standard Parts.</li><br />
<li>Designed parts in conformity with accepted BioBrick standards.</li><br />
<li>DNA for 49 BioBricks entered in the Registry were sent in to iGEM headquarters. The majority of these parts were confirmed with DNA sequencing.</li><br />
</ul><br />
<br><br />
<br />
<a name="silver"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Silver Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Demonstrated that several submitted BioBricks work as expected.</li><br />
<p class="margin">- The composite lac promoter/RBS/GFP/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208045"><b><font color=#009900>BBa_K208045</font></b></a>). This composite was demonstrated by the presence of GFP visualized on a UV transilluminator. The GFP protein was also visualized using SDS polyacrylamide gel electrophoresis. See pictures on Wiki. </p> <br />
<p class="margin">- New GFP reporter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208000"><b><font color=#009900>BBa_K208000</font></b></a>). This reporter, with an excitation/emission of 395/509, was shown to be functional using the novel composite construct BBa_K2208045 (lac promoter/RBS/GFP/terminator). Picture on Wiki under part BBa_K2208045. </p><br />
<p class="margin">- The composite lac promoter/RBS (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010"><b><font color=#009900>BBa_K208010</font></b></a>). This was demonstrated using the composite construct BBa_K2208045. Picture on Wiki under part BBa_K2208045. This composite part is extremely useful because it alleviates the need to work with extremely small ribosomal BioBrick components. </p> <br />
<p class="margin">- Lac promoter/RBS/Gene III/Phasin/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208038"><b><font color=#009900>BBa_K208038</font></b></a>). This was demonstrated through the detection of phasin proteins isolated from supernatant samples using SDS polyacrylamide gel electrophoresis. Picture on Wiki. </p><br />
<p class="margin">- Phasin gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208001"><b><font color=#009900>BBa_K208001</font></b></a>). A protein of the correct size was detected in SDS polyacrylamide gel electrophoresis from cells having this construct that was not detected in the control samples. <br />
<p class="margin">- Gene III secretion tag (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208002"><b><font color=#009900>BBa_K208002</font></b></a>). The phasin protein from part BBa_K208038 was detected outside the cell, thus demonstrating the functionality of this Gene III secretion tag.</p><br />
<li>Characterized the GFP reporter (BBa_K208000) (1) showing GFP expression over time and (2) showing sensitivity of IPTG induction.</li><br />
</ul><br><br />
<br />
<a name="gold"></a><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Gold Medal Requirements</b></font><br />
<Br><br />
<ul class="circle"><br />
<li>Improved upon the existing set of GFP BioBrick parts by constructing a GFP with a larger Stokes shift (395/509nm), making for easier downsteam analysis. The bright fluorescence can very easily be seen on a standard UV transilluminator. As GFP is one of the most commonly used reporters, the introduction of this new and improved BioBrick part should be of great benefit to the iGEM community.</li> <br />
<li>Our initial plans for our 2009 team was in part to continue the 2008 Hawaii team’s project, “Cyanobacteria Toolkit,” by making the pRL1383a vector an effective broad-host vector. When this vector proved ineffective, we made efforts to troubleshoot. We had much correspondence with the advisors of the Hawaii team, including a conference call mid-summer. Since most of their parts were not included in the 2009 iGEM distribution, we had them send many of the parts they used. After numerous approaches and attempts to convert pRL1383a into BioBrick format, we finally decided that the vector was either mischaracterized or altered in some unknown way. </li> <br />
<li>As thoroughly explained in our Wiki, one of the primary goals of our project has been to expand the BioBrick world to organisms other than E. coli. We have documented the many benefits that would come from such an achievement. Though the pRL1383a vector efforts were unsuccessful, we did successfully express the vector pCPP33 in E. coli, Pseudomonas putida, Synechocystis pcc 6803, and Rhodobacter sphaeroides. This vector now has only to be put in BioBrick format. </li> <br />
<li>In reference to our ethics section (https://2009.igem.org/Team:Utah_State/ETHICS), our team has taken specific measures to follow our suggested proposals. In the education of our team, we discussed the potential benefits of a standard secretion system but also discussed the potential of our designed secretion pathways to be used in a malevolent manner. As a team, we acknowledge the importance of high moral accountability and commitment to safety and security. Additionally, in an effort to foster the sharing of information in our community, upon completion of the jamboree we will submit an article to be released in our school newspaper and in the College of Engineering website. This winter we are also hosting a lecture by Drew Endy addressing synthetic biology which will be open to the public. We hope to act as ambassadors to foster support and excitement in our own community.</li> <br />
<br />
</td><br />
</tr><br />
</tr><br />
</table><br />
</html><br />
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</html></div>Liblinthttp://2009.igem.org/Team:Utah_State/AchievementsTeam:Utah State/Achievements2009-11-12T04:13:41Z<p>Liblint: </p>
<hr />
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<table width="100%" border="0"><br />
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<td width="16%" valign="top"><table width="72%" border="0"><br />
<tr valign="top"><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>JUDGING</font></span><br />
<a href="#bronze">Bronze</a><br /><br />
<a href="#silver">Silver</a><br /><br />
<a href="#gold">Gold</a><br /><br />
</td> <br />
</tr><br />
<br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
</tr><br />
</table></td><br />
<td><table width=100% style="background:#CCCCCC; padding:7px; border-style:none"><br />
<tr><br />
<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>JUDGING CRITERIA</b></i></font><br />
<HR><br />
<br />
<font size="3" face="Helvetica, Arial, San Serif" color =#231f20><b><i>In fulfillment of the requirements for the Gold Medal, the 2009 Utah State iGEM Team did the following:</b></i></font><br><br />
<br><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Bronze Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Completed the registration requirements and Project Summary form.</li><br />
<li>Prepared and will present a poster and talk at the 2009 Jamboree.</li><br />
<li>Entered all necessary information detailing 62 BioBricks into the Registry of Standard Parts.</li><br />
<li>Designed parts in conformity with accepted BioBrick standards.</li><br />
<li>DNA for 49 BioBricks entered in the Registry were sent in to iGEM headquarters. The majority of these parts were confirmed with DNA sequencing.</li><br />
</ul><br />
<br><br />
<br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Silver Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Demonstrated that several submitted BioBricks work as expected.</li><br />
<p class="margin">- The composite lac promoter/RBS/GFP/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208045"><b><font color=#009900>BBa_K208045</font></b></a>). This composite was demonstrated by the presence of GFP visualized on a UV transilluminator. The GFP protein was also visualized using SDS polyacrylamide gel electrophoresis. See pictures on Wiki. </p> <br />
<p class="margin">- New GFP reporter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208000"><b><font color=#009900>BBa_K208000</font></b></a>). This reporter, with an excitation/emission of 395/509, was shown to be functional using the novel composite construct BBa_K2208045 (lac promoter/RBS/GFP/terminator). Picture on Wiki under part BBa_K2208045. </p><br />
<p class="margin">- The composite lac promoter/RBS (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010"><b><font color=#009900>BBa_K208010</font></b></a>). This was demonstrated using the composite construct BBa_K2208045. Picture on Wiki under part BBa_K2208045. This composite part is extremely useful because it alleviates the need to work with extremely small ribosomal BioBrick components. </p> <br />
<p class="margin">- Lac promoter/RBS/Gene III/Phasin/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208038"><b><font color=#009900>BBa_K208038</font></b></a>). This was demonstrated through the detection of phasin proteins isolated from supernatant samples using SDS polyacrylamide gel electrophoresis. Picture on Wiki. </p><br />
<p class="margin">- Phasin gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208001"><b><font color=#009900>BBa_K208001</font></b></a>). A protein of the correct size was detected in SDS polyacrylamide gel electrophoresis from cells having this construct that was not detected in the control samples. <br />
<p class="margin">- Gene III secretion tag (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208002"><b><font color=#009900>BBa_K208002</font></b></a>). The phasin protein from part BBa_K208038 was detected outside the cell, thus demonstrating the functionality of this Gene III secretion tag.</p><br />
<li>Characterized the GFP reporter (BBa_K208000) (1) showing GFP expression over time and (2) showing sensitivity of IPTG induction.</li><br />
</ul><br><br />
<br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Gold Medal Requirements</b></font><br />
<Br><br />
<ul class="circle"><br />
<li>Improved upon the existing set of GFP BioBrick parts by constructing a GFP with a larger Stokes shift (395/509nm), making for easier downsteam analysis. The bright fluorescence can very easily be seen on a standard UV transilluminator. As GFP is one of the most commonly used reporters, the introduction of this new and improved BioBrick part should be of great benefit to the iGEM community.</li> <br />
<li>Our initial plans for our 2009 team was in part to continue the 2008 Hawaii team’s project, “Cyanobacteria Toolkit,” by making the pRL1383a vector an effective broad-host vector. When this vector proved ineffective, we made efforts to troubleshoot. We had much correspondence with the advisors of the Hawaii team, including a conference call mid-summer. Since most of their parts were not included in the 2009 iGEM distribution, we had them send many of the parts they used. After numerous approaches and attempts to convert pRL1383a into BioBrick format, we finally decided that the vector was either mischaracterized or altered in some unknown way. </li> <br />
<li>As thoroughly explained in our Wiki, one of the primary goals of our project has been to expand the BioBrick world to organisms other than E. coli. We have documented the many benefits that would come from such an achievement. Though the pRL1383a vector efforts were unsuccessful, we did successfully express the vector pCPP33 in E. coli, Pseudomonas putida, Synechocystis pcc 6803, and Rhodobacter sphaeroides. This vector now has only to be put in BioBrick format. </li> <br />
<li>In reference to our ethics section (https://2009.igem.org/Team:Utah_State/ETHICS), our team has taken specific measures to follow our suggested proposals. In the education of our team, we discussed the potential benefits of a standard secretion system but also discussed the potential of our designed secretion pathways to be used in a malevolent manner. As a team, we acknowledge the importance of high moral accountability and commitment to safety and security. Additionally, in an effort to foster the sharing of information in our community, upon completion of the jamboree we will submit an article to be released in our school newspaper and in the College of Engineering website. This winter we are also hosting a lecture by Drew Endy addressing synthetic biology which will be open to the public. We hope to act as ambassadors to foster support and excitement in our own community.</li> <br />
<br />
</td><br />
</tr><br />
</tr><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>HOME</font></span><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
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<b><i>Welcome!</b></i></font><br />
<HR><br />
<p class="class"><br />
The Utah State University team is happy to have received a GOLD MEDAL at the 2009 iGEM Jamboree. We are very proud of all that we were able to accomplish this year. We invite you to explore our site and learn all about our project! And please contact us if you would like more information about any aspect of our project.</p><br />
<p class = "class"> The aim of the Utah State University iGEM project is to develop improved production and harvesting methods of proteins and other products in multiple organisms using the standardized BioBrick system. The name of our project, BioBricks without Borders, characterizes and ties together the two main focuses of our research:</p></br><br />
<ul class="circle"><br />
<li>Investigating broad host-range vectors for production of compounds in organisms other than <i>E. coli</i> (like <i>Synechocystis</i> PCC6803, <i>Rhodobacter sphaeroides</i>, and <i>Pseduomonas putida</i>)<br />
<li>Developing a library of fusion-compatible BioBrick parts for targeting compounds for secretion</li><br />
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<a href="http://www.bie.usu.edu/"><img src="https://static.igem.org/mediawiki/2009/c/c8/Bio_Engineering-04.png" align = "middle" height="92" style="float:center;" alt="BIE"></a><br />
<a href="http://www.usu.edu/"><img src="https://static.igem.org/mediawiki/2009/d/db/UsuLogo_BLWboard-02.png" align = "middle" height="92" style="float:center;" alt="USU"> </div><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>CONTACT</font></font></span><br />
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<b><i>Contact Us</b></i></font><br />
<HR><br />
<p class="class"><br />
We would like to hear your questions and comments! Please send us an email and come talk to us at the Jamboree! Thanks for your interest in USU iGEM 2009. Be sure to also check out the links to our sponsors web pages - we have greatly appreciated their help. </p><br />
<br />
<ul class="circle"><br />
<li>Dr. Charles Miller: charles.miller@engineering.usu.edu </li><br />
<li>The USU iGEM Team: usu.igem.2009@gmail.com </li></ul><br />
<br />
</td><br />
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<p class="class"><br />
Our team would also like to thank Erika Johnsen for her help with the USU iGEM team logo. Follow the link below to check out her website for more information. </p><br />
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<br />
<a href="http://www.erikajohnsendesign.com/"><img src="https://static.igem.org/mediawiki/igem.org/b/b8/Erikajohnsen.png" align = "middle" height="30" alt="EJ"> </a><br />
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<a href="http://www.bie.usu.edu/"><img src="https://static.igem.org/mediawiki/2009/c/c8/Bio_Engineering-04.png" align = "middle" height="92" style="float:center;" alt="BIE"></a><br />
<a href="http://www.usu.edu/"><img src="https://static.igem.org/mediawiki/2009/d/db/UsuLogo_BLWboard-02.png" align = "middle" height="92" style="float:center;" alt="USU"> </div><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
</tr><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>CONTACT</font></font></span><br />
</tr><br />
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<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>Contact Us</b></i></font><br />
<HR><br />
<p class="class"><br />
We would like to hear your questions and comments! Please send us an email and come talk to us at the Jamboree! Thanks for your interest in USU iGEM 2009. Be sure to also check out the links to our sponsors web pages - we have greatly appreciated their help. </p><br />
<br />
<ul class="circle"><br />
<li>Dr. Charles Miller: charles.miller@engineering.usu.edu </li><br />
<li>The USU iGEM Team: usu.igem.2009@gmail.com </li></ul><br />
<br />
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<a href="http://www.bie.usu.edu/"><img src="https://static.igem.org/mediawiki/2009/c/c8/Bio_Engineering-04.png" align = "middle" height="92" style="float:center;" alt="BIE"></a><br />
<a href="http://www.usu.edu/"><img src="https://static.igem.org/mediawiki/2009/d/db/UsuLogo_BLWboard-02.png" align = "middle" height="92" style="float:center;" alt="USU"> </div><br />
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</html></div>Liblinthttp://2009.igem.org/Team:Utah_State/AchievementsTeam:Utah State/Achievements2009-10-22T03:51:51Z<p>Liblint: </p>
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<table width="100%" border="0"><br />
<tr><br />
<td width="16%" valign="top"><table width="72%" border="0"><br />
<tr valign="top"><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>JUDGING</font></span><br />
<a href="#bronze">Bronze</a><br /><br />
<a href="#silver">Silver</a><br /><br />
<a href="#gold">Gold</a><br /><br />
</td> <br />
</tr><br />
<br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
</tr><br />
</table></td><br />
<td><table width=100% style="background:#CCCCCC; padding:7px; border-style:none"><br />
<tr><br />
<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>JUDGING CRITERIA</b></i></font><br />
<HR><br />
<br />
<font size="3" face="Helvetica, Arial, San Serif" color =#231f20><b><i>In fulfillment of the requirements for the Gold Medal, the 2009 Utah State iGEM Team did the following:</b></i></font><br><br />
<br><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Bronze Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Completed the registration requirements and Project Summary form.</li><br />
<li>Prepared and will present a poster and talk at the 2009 Jamboree.</li><br />
<li>Entered all necessary information detailing 62 BioBricks into the Registry of Standard Parts.</li><br />
<li>Designed parts in conformity with accepted BioBrick standards.</li><br />
<li>DNA for 49 BioBricks entered in the Registry were sent in to iGEM headquarters. The majority of these parts were confirmed with DNA sequencing.</li><br />
</ul><br />
<br><br />
<br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Silver Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Demonstrated that several submitted BioBricks work as expected.</li><br />
<p class="margin">- The composite lac promoter/RBS/GFP/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208045"><b><font color=#009900>BBa_K2208045</font></b></a>). This composite was demonstrated by the presence of GFP visualized on a UV transilluminator. The GFP protein was also visualized using SDS polyacrylamide gel electrophoresis. See pictures on Wiki. </p> <br />
<p class="margin">- New GFP reporter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208000"><b><font color=#009900>BBa_K2208000</font></b></a>). This reporter, with an excitation/emission of 395/509, was shown to be functional using the novel composite construct BBa_K2208045 (lac promoter/RBS/GFP/terminator). Picture on Wiki under part BBa_K2208045. </p><br />
<p class="margin">- The composite lac promoter/RBS (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010"><b><font color=#009900>BBa_K2208010</font></b></a>). This was demonstrated using the composite construct BBa_K2208045. Picture on Wiki under part BBa_K2208045. This composite part is extremely useful because it alleviates the need to work with extremely small ribosomal BioBrick components. </p> <br />
<p class="margin">- Lac promoter/RBS/Gene III/Phasin/terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208038"><b><font color=#009900>BBa_K2208038</font></b></a>). This was demonstrated through the detection of phasin proteins isolated from supernatant samples using SDS polyacrylamide gel electrophoresis. Picture on Wiki. </p><br />
<p class="margin">- Phasin gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208001"><b><font color=#009900>BBa_K2208001</font></b></a>). A protein of the correct size was detected in SDS polyacrylamide gel electrophoresis from cells having this construct that was not detected in the control samples. <br />
<p class="margin">- Gene III secretion tag (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208002"><b><font color=#009900>BBa_K2208002</font></b></a>). The phasin protein from part BBa_K208038 was detected outside the cell, thus demonstrating the functionality of this Gene III secretion tag.</p><br />
<li>Characterized the GFP reporter (BBa_K208000) (1) showing GFP expression over time and (2) showing sensitivity of IPTG induction.</li><br />
</ul><br><br />
<br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Gold Medal Requirements</b></font><br />
<Br><br />
<ul class="circle"><br />
<li>Improved upon the existing set of GFP BioBrick parts by constructing a GFP with a larger Stokes shift (395/509nm), making for easier downsteam analysis. The bright fluorescence can very easily be seen on a standard UV transilluminator. As GFP is one of the most commonly used reporters, the introduction of this new and improved BioBrick part should be of great benefit to the iGEM community.</li> <br />
<li>Our initial plans for our 2009 team was in part to continue the 2008 Hawaii team’s project, “Cyanobacteria Toolkit,” by making the pRL1383a vector an effective broad-host vector. When this vector proved ineffective, we made efforts to troubleshoot. We had much correspondence with the advisors of the Hawaii team, including a conference call mid-summer. Since most of their parts were not included in the 2009 iGEM distribution, we had them send many of the parts they used. After numerous approaches and attempts to convert pRL1383a into BioBrick format, we finally decided that the vector was either mischaracterized or altered in some unknown way. </li> <br />
<li>As thoroughly explained in our Wiki, one of the primary goals of our project has been to expand the BioBrick world to organisms other than E. coli. We have documented the many benefits that would come from such an achievement. Though the pRL1383a vector efforts were unsuccessful, we did successfully express the vector pCPP33 in E. coli, Pseudomonas putida, Synechocystis pcc 6803, and Rhodobacter sphaeroides. This vector now has only to be put in BioBrick format. </li> <br />
<li>In reference to our ethics section (https://2009.igem.org/Team:Utah_State/ETHICS), our team has taken specific measures to follow our suggested proposals. In the education of our team, we discussed the potential benefits of a standard secretion system but also discussed the potential of our designed secretion pathways to be used in a malevolent manner. As a team, we acknowledge the importance of high moral accountability and commitment to safety and security. Additionally, in an effort to foster the sharing of information in our community, upon completion of the jamboree we will submit an article to be released in our school newspaper and in the College of Engineering website. This winter we are also hosting a lecture by Drew Endy addressing synthetic biology which will be open to the public. We hope to act as ambassadors to foster support and excitement in our own community.</li> <br />
<br />
</td><br />
</tr><br />
</tr><br />
</table><br />
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</html></div>Liblinthttp://2009.igem.org/Team:Utah_State/AchievementsTeam:Utah State/Achievements2009-10-22T03:47:19Z<p>Liblint: </p>
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<div><html><br />
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<tr valign="top"><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>JUDGING</font></span><br />
<a href="#bronze">Bronze</a><br /><br />
<a href="#silver">Silver</a><br /><br />
<a href="#gold">Gold</a><br /><br />
</td> <br />
</tr><br />
<br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
</tr><br />
</table></td><br />
<td><table width=100% style="background:#CCCCCC; padding:7px; border-style:none"><br />
<tr><br />
<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>JUDGING CRITERIA</b></i></font><br />
<HR><br />
<br />
<font size="3" face="Helvetica, Arial, San Serif" color =#231f20><b><i>In fulfillment of the requirements for the Gold Medal, the 2009 Utah State iGEM Team did the following:</b></i></font><br><br />
<br><br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Bronze Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Completed the registration requirements and Project Summary form.</li><br />
<li>Prepared and will present a poster and talk at the 2009 Jamboree.</li><br />
<li>Entered all necessary information detailing 62 BioBricks into the Registry of Standard Parts.</li><br />
<li>Designed parts in conformity with accepted BioBrick standards.</li><br />
<li>DNA for 49 BioBricks entered in the Registry were sent in to iGEM headquarters. The majority of these parts were confirmed with DNA sequencing.</li><br />
</ul><br />
<br><br />
<br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Silver Medal Requirements</b></font><br />
<ul class="circle"><br />
<li>Demonstrated that several submitted BioBricks work as expected.</li><br />
<p class="margin">- The composite lac promoter/RBS/GFP/terminator (BBa_K2208045). This composite was demonstrated by the presence of GFP visualized on a UV transilluminator. The GFP protein was also visualized using SDS polyacrylamide gel electrophoresis. See pictures on Wiki. </p> <br />
<p class="margin">- New GFP reporter (BBa_K208000). This reporter, with an excitation/emission of 395/509, was shown to be functional using the novel composite construct BBa_K2208045 (lac promoter/RBS/GFP/terminator). Picture on Wiki under part BBa_K2208045. </p><br />
<p class="margin">- The composite lac promoter/RBS (BBa_K208010). This was demonstrated using the composite construct BBa_K2208045. Picture on Wiki under part BBa_K2208045. This composite part is extremely useful because it alleviates the need to work with extremely small ribosomal BioBrick components. </p> <br />
<p class="margin">- Lac promoter/RBS/Gene III/Phasin/terminator (BBa_K208038). This was demonstrated through the detection of phasin proteins isolated from supernatant samples using SDS polyacrylamide gel electrophoresis. Picture on Wiki. </p><br />
<p class="margin">- Phasin gene (BBa_K208001). A protein of the correct size was detected in SDS polyacrylamide gel electrophoresis from cells having this construct that was not detected in the control samples. <br />
<p class="margin">- Gene III secretion tag (BBa_K208002). The phasin protein from part BBa_K208038 was detected outside the cell, thus demonstrating the functionality of this Gene III secretion tag.</p><br />
<li>Characterized the GFP reporter (BBa_K208000) (1) showing GFP expression over time and (2) showing sensitivity of IPTG induction.</li><br />
</ul><br><br />
<br />
<font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><b>Gold Medal Requirements</b></font><br />
<Br><br />
<ul class="circle"><br />
<li>Improved upon the existing set of GFP BioBrick parts by constructing a GFP with a larger Stokes shift (395/509nm), making for easier downsteam analysis. The bright fluorescence can very easily be seen on a standard UV transilluminator. As GFP is one of the most commonly used reporters, the introduction of this new and improved BioBrick part should be of great benefit to the iGEM community.</li> <br />
<li>Our initial plans for our 2009 team was in part to continue the 2008 Hawaii team’s project, “Cyanobacteria Toolkit,” by making the pRL1383a vector an effective broad-host vector. When this vector proved ineffective, we made efforts to troubleshoot. We had much correspondence with the advisors of the Hawaii team, including a conference call mid-summer. Since most of their parts were not included in the 2009 iGEM distribution, we had them send many of the parts they used. After numerous approaches and attempts to convert pRL1383a into BioBrick format, we finally decided that the vector was either mischaracterized or altered in some unknown way. </li> <br />
<li>As thoroughly explained in our Wiki, one of the primary goals of our project has been to expand the BioBrick world to organisms other than E. coli. We have documented the many benefits that would come from such an achievement. Though the pRL1383a vector efforts were unsuccessful, we did successfully express the vector pCPP33 in E. coli, Pseudomonas putida, Synechocystis pcc 6803, and Rhodobacter sphaeroides. This vector now has only to be put in BioBrick format. </li> <br />
<li>In reference to our ethics section (https://2009.igem.org/Team:Utah_State/ETHICS), our team has taken specific measures to follow our suggested proposals. In the education of our team, we discussed the potential benefits of a standard secretion system but also discussed the potential of our designed secretion pathways to be used in a malevolent manner. As a team, we acknowledge the importance of high moral accountability and commitment to safety and security. Additionally, in an effort to foster the sharing of information in our community, upon completion of the jamboree we will submit an article to be released in our school newspaper and in the College of Engineering website. This winter we are also hosting a lecture by Drew Endy addressing synthetic biology which will be open to the public. We hope to act as ambassadors to foster support and excitement in our own community.</li> <br />
<br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br /><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Introduction: Why Break BioBrick Borders?<br />
</font></b></i> <hr><br />
<p class = "class"><br />
Since the beginning of iGEM, BioBricks have chiefly been designed for use in <i>E.coli</i>. This has primarily been due to the efficient growth rate of <i>E.coli</i> and its relatively thorough characterization. However, the employment of the BioBrick system in host organisms other than <i>E. coli</i> would greatly enhance and expand the field of synthetic biology. In order to investigate the BioBrick system in other organisms, it is imperative that a reliable broad-host-range vector be developed. The 2009 Utah State iGEM team is building on the 2008 University of Hawaii team’s efforts to develop a broad-host-range BioBrick vector that would make possible the use of BioBrick parts, devices, and systems in organisms other than <i>E. coli</i>. The organisms under investigation are <i>Pseudomonas putida</i> KT2440, <i>Synechocystis</i> PCC 6803, and <i>Rhodobacter sphaeroides</i>. Additionally, our project seeks to break borders in another way: through the construction of Silver-fusion compatible BioBrick parts for secretion-based recovery of recombinant proteins and other compounds, like polyhydroxyalkanoates. </p><br />
<br />
<p class="class"><br />
One of the BioBrick borders we seek to break is that of <i>Pseudomonas putida</i>. This bacterium would open the BioBrick doors to soil applications. <i>Pseudomonas putida</i> is a non-pathogenic, gram-negative soil bacterium with optimal growth at room temperature. The diversity of its metabolic pathways allows it to be used for bioremediation purposes; it can degrade many polluting aromatic hydrocarbons including toluene, benzene, xylene, naphthalene, and styrene. This organism can also act as a biocontrol agent (Lemanceau, 1992; Haas and Defago, 2005), suppressing the growth of fungi. <i>P. putida</i> performs these functions while colonizing the rhizosphere of plant roots, enhancing the growth of the plant through these and other means (Albert and Anderson, 1987; Bakker et al., 1986). The genome of <i>P. putida</i> KT2440 has been sequenced, allowing more extensive genetic analyses and contributing to this strain being the “preferred host for cloning and gene expression for Gram-negative soil bacteria” (Nelson et al., 2002). Potential applications include BioBrick devices for enhancing the catabolism of environmental pollutants, the implementation of BioBrick devices used to protect plants (and its subsequent consumers) against pathogens not previously defended against, and the use of BioBrick devices to increase crop yields. </p><br />
<br />
<p class="class"><br />
Another BioBrick border we'd like to break is that of cyanobacteria. We have specifically been working with <i>Synechocystis</i> PCC 6803. This bacterium would allow BioBricks to be used in photosynthetic applications. <i>Synechocystis</i> PCC 6803 is a Gram-negative bacterium that can produce energy either through photosynthesis or respiration (Tabei et al., 2007). It also displays a circadian rhythm in several of its cellular functions (Kucho et al., 2005) and can take up foreign DNA (Williams 1988). It can also grow in a variety of temperatures (Gombos et al., 1992). Cyanobacteria in general play an important role in nitrogen fixation for crops and are a major player in rice cultivation (Irisarri et al., 2001). Potential applications include the use of BioBrick devices in bioenergy, wastewater treatment, crop yields, and biomanufacturing processes that take advantage of the fact that a carbon source is not needed.</p><br />
<br />
<p class="class"> A third border that we aim to break is that of <i>Rhodobacter sphaeroides</i>, an organism usually found in the anaerobic mud of ponds and lakes where there is access to sunlight. This is a very metabolically diverse organism that has potential for providing a myriad of BioBrick opportunities. <i>Rhodobacter sphaeroides</i> can grow under a variety of conditions: aerobic or anaerobic respiration, photosynthesis, and fermentation; it has optimal growth in microaerophilic surroundings. It can also fix dinitrogen as its sole nitrogen source (Mackenzie et al., 2007). Similar to E. coli, this organism moves with a single flagellum. <i>R. sphaeroides</i> has more membrane surface per cell than other organisms used to express membrane proteins, making it an ideal host for overexpressing and studying such proteins. It is capable of making biofuels through the process of lithotrophy (Roy et al., 2008) and other pathways (Yokoi et al., 2002). <i>R. sphaeroides</i> is also capable of tolerating and reducing at least 11 rare earth metal oxides and oxyanions, making it an excellent candidate for bioremediation and detoxification purposes (O’Gara et al., 1997). Many of the above-listed characteristics place <i>R. sphaeroides</i> in the spotlight for use in biomanufacturing. Possible BioBrick applications with <i>R. sphaeroides</i> include membrane protein studies (including secretion and protein overexpression studies), biomanufacturing, bioenergy, and bioremediation/detoxification. </p><br />
<br />
<p class="class">To <i>even further</i> break down BioBrick borders, composite devices were constructed to investigate phasin and green fluorescent protein secretion. Secretion of phasin was studied to show that these PHA-associated proteins are targetable for export out of the cytoplasm, and that optimization of phasin expression and binding may facilitate bioplastic secretion. Constructs for GFP translocation were made in parallel with the phasin secretion devices. These GFP constructs provide a visually or spectrofluorometrically detectable control due to a high level of fluorescent protein accumulation. Successful GFP translocation would reinforce the potential of phasin export, which is not as readily monitored. Beyond the scope of this project, the constructed signal peptides and GFP BioBricks can readily be used by other researchers for recombinant protein secretion studies.</p><br />
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<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Project Objectives<br />
</font></b></i> <br />
<p class = "class">The overall goal of this project is to demonstrate the concept of “BioBricks without Borders” by expanding the use of broad-host vectors for expression of BioBricks in multiple organisms and by demonstrating secretion for simplified recovery of recombinant proteins using BioBrick constructs. More specific goals of this project are to:</p><br />
<br />
<ul class= "circle"><br />
<li>Determine how broad-host range vectors can be modified to comply with the BioBrick assembly standard.</li><br />
<li>Use broad-host range vectors to transform <i>Synechocystis</i> PCC6803, <i>R. sphaeroides</i>, and <i>P. putida</i> by triparental mating. </li><br />
<li>Create a BioBrick genetic library of Silver fusion-compatible signal peptides and coding regions for secretion studies.</li><br />
<li>Test the functionality of BioBrick devices and determine methods for detecting phasin and/or PHA secretion.</li><br />
</ul><br />
<br />
<p class = "class">The following sections provide more extensive details about these goals, experimentation and testing, and the results and conclusions from this project.</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/Broad-HostVectorsTeam:Utah State/Broad-HostVectors2009-10-22T03:32:12Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br /><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Experiments"><font size = 4>EXPERIMENTS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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Broad-Host Range Vectors<br />
</font></b></i> <hr><br />
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<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Advantages for Using a Broad Host Range Vector<br />
</font></b></i> <br />
<br><br />
<p class="class">A multi-host vector allows for genetic manipulation to occur in one organism, and the ultimate application of the vector to be served in another. Genetic manipulation is ideally done in <i>E. coli</i>, due to its fast growth, ease of use, and availability of transformable cells. However, it does not always represent the best choice for production of recombinant proteins or other compounds, and thus it is ideal to be able to transfer genetic information into other organisms once manipulation and testing of the construct is complete.</p><br />
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<p class="class">Most broad host range vectors are naturally occurring or a derivative of a natural vector. They tend to be large, around 10 kbp, although some commercial versions have been optimized to a much shorter length (http://www.bio101.com/functional-analysis/pBBR122.html). They can be self-transmissible (presence of <i>tra</i> genes) and mobilizable (mob genes), but desirable vectors are both mobilizable and non-transmissible (Haller, & Dichristina, 2002). This allows for more control over conjugation in the laboratory through use of a helper plasmid (Haller, & Dichristina, 2002). A helper plasmid is a conjugative plasmid, that is it contains both transmission and mobilization genes. While a broad-host range plasmid can be conjugated into another organism, its copy number will remain undetectably low unless a fully functioning helper plasmid is present (Haller, & Dichristina, 2002).. If a helper plasmid shares the same origin of transfer (oriT), mob genes are no longer necessary (Snyder and Champness 2007). Due to this property, the mob genes of commercial plasmids are often removed, thereby resulting in vectors that are significantly shorter than their natural counterparts (Snyder and Champness 2007). Use of a helper plasmid becomes necessary if the self-transmission genes are not present to achieve any detectable degree of replication in the recipient organism (Haller, & Dichristina, 2002).</p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#000033><br />
Genetic Characteristics of Broad-Host Range Vectors<br />
</font></b></i><br />
<br />
<p class="class">Broad host range vectors are a class of mobilizable plasmids, that is they lack the complete tra-genes necessary for conjugation but can still transfer and replicate at high copy number in the presence of a conjugative plasmid. Mobilizable vectors still contain some of the genes necessary for transfer. The mob genes code proteins that aid the vector in transferring from one organism to another. One protein produced in the region, nickase-helicase, nics the DNA at the origin of transfer (oriT). As the envelopes of the two cells meet, the mobility proteins synthesize a new strand of DNA from the plasmid parent strand as it enters the recipient cell. A new strand is also synthesized in the donor cell simultaneously. In this way, the plasmid is transferred from one cell to another (Porter, 2002).</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/Mechanism_of_Bacerial_Conjugation.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 1.</b> Mechanism for bacterial conjugation<br />
</div><br />
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<p class="class">The multi-host vector pRL1383a was used in this study. It is derived from RSF1010, a naturally occurring broad host range vector found in <i>E. coli</i>. RSF 1010 has been completely sequenced (Scholz 1989). It is designed for use in Cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Two versions of this vector were tested: one containing mob A/B/C genes with an origin of transfer (Figure 2), and one utilizing an RP4 origin of transfer (matching the origin of transfer in the RP4 helper plasmid. This eliminates the need for mobilization genes when used with this helper vector). In addition, the vector has resistance cassettes for both Streptomycin and Spectinomycin (Wolk 2007).</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/SecretionTeam:Utah State/Secretion2009-10-22T03:31:08Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a> <br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br /><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. <i>coli</i> MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. <i>coli</i> transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. <i>coli</i> or photoautotrophic PHA-producers R. <i>sphaeroides</i> and <i>Synechocystis</i> PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure 1 depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 1.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant <i>E. coli</i> do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure 2 depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. <i>coli</i> K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure 3. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative E. <i>coli</i> (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure 3 illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 3.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure 4 shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 4.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of E. <i>coli</i> (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in E. <i>coli</i> harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in E. <i>coli</i> (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 5 depicts the GFP barrel structure.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="200" style="padding:.5px; alt="signal peptide" /> </div><br />
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<b>Figure 5.</b> The GFP Barrel Structure<br />
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<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. <i>coli</i>, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
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<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
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References<br />
</font></b></i> <hr><br />
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<li>Veenendaal AKL, van der Does C, Driessen AJM (2004) The protein-conducting channel SecYEG. Biochimica et Biophysica Act 1694:81-95</li><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br /><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Experimental Section: Approach for BioBrick Compatibility<br />
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Converting Broad-Host Vectors into a BioBrick-Compatible Format<br />
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<p class="class">Two Broad-host range vectors were used in this study; pRL1383a and PCPP33. To convert these vectors into BioBrick-compatible format, the four standard BioBrick sites EcoRI, XbaI, SpeI, and PstI needed to be inserted into the multiple cloning site. For pRL1383a, common BioBrick primers VR and VF2 were also included to allow the use of PCR in amplifying the BioBrick parts.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/8/82/PRL1383A_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 2</b> Plasmid map of pRL1383a <br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/PCPP33_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 3</b> Plasmid map of pCPP33 <br />
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<p class="class">Apart from being shown effective in the Synechosystis PCC 6803 (Marraccini 1993), pRL1383a is an ideal candidate for use as a BioBrick-compatible broad-host range vector because the BioBrick restriction sites are absent within the vector sequence. To convert pRL1383a into a BioBrick format, the existing multiple cloning site, which is flanked by a BamHI site and a HindIII site, was utilized. First, modified primers were synthesized from BioBrick primers VR and VF2. These primers were modified by adding extra nucleotides to insert the desired restriction enzyme sites into the PCR product. A BamHI site was added to 5’ end of the forward primer (VF2) and a HindIII site was added to the 5’ end of the reverse primer (VR). These primers were used to amplify an existing, tested BioBrick part by PCR. For this purpose, we selected BBa_I20260 because it does not contain BamHI or HindIII sites, and successful ligation is readily testable as it is a GFP -producing construct. The addition of IPTG is typically necessary to induce GFP production in this particular device. However, when using Top10 <i>E. coli</i> cells it is produced continuously because these cells lack a lac repressor (insert invitrogen link). After cutting the vector at the multiple cloning site using BamHI and HindIII, the BioBrick insert obtained by PCR with modified ends was ligated into the backbone. The vector was then transformed using Top10 One Shot® chemically competent <i>E. coli</i> and tested for successful insertion using PCR and restriction digests.</p><br />
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<p class="class">Another broad host range vector, pCPP33, previously shown effective in Pseudomonas Putida,was standardized using similar methods. While the complete sequence of this plasmid is not available, it was shown that there are no BioBrick restriction sites outside the multiple cloning site (Figure 3). The multiple cloning site of this vector is flanked by EcoRI and HindIII. This allowed the PCR product of BBa_I20260 to again be used by cutting with HindIII and EcoRI restriction enzymes. Restriction digests and gel analysis were used to test for the insert.</p><br />
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Broad Host Conjugation<br />
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<p class="class">In order to transfer a vector of interest using conjugation, the <i>tra</i> gene (contained in what we will refer to as a transfer plasmid, or helper plasmid) must be expressed in order to initiate the conjugation process. This plasmid codes for genes which, when expressed, form pili on the cell surface, which in turn initiate conjugation (Heinemann 1989). This plasmid may be present in one of three different procedures:</p><br />
<ul><br />
</li><li><b>Hfr strain</b> – The <i>tra</i> operon is many times contained in an episome, which can incorporate itself into the cell genome. These resultant Hfr strains will often begin the transfer of their own DNA, both plasmid and genomic. Due to the transfer of the genomic DNA, these strains are referred to as high frequency recombinant (Hfr) strains.<br />
<br />
</li><li><b>Biparental (normal) Conjugation</b> – Cells containing the <i>tra</i> genes, often labeled as F-positive (F+) due to the F-plasmid, a well-known transfer plasmid, can express the transfer genes necessary for conjugation to occur. When a vector of interest and a transfer plasmid are of different incompatibility groups, they may both be transformed into the same cell, and conjugation may take place between the F+ donor cell and the recipient cell<br />
<br />
</li><li><b>Triparental Mating</b> – In the case where the transfer plasmid and the vector of interest are of the same incompatibility group, the two plasmids may not stably coexist (Heinemann 1989). In this case, two separate cells containing the transfer gene (the helper cell) and the vector (the donor cell) must be used in conjugation. The helper cell will assist the donor cell in the transfer of its mobilizable plasmid to the recipient cell. This method circumvents some of the barriers that may prevent the transfer of plasmids.<br />
</ul><br />
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<br />
<p class="class">For our project, we chose to use the triparental mating procedure for the transmission of our vector. While not being the most efficient method, it circumvents possible barriers and intermediate steps.</p><br />
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<p class="class">Because of the use of three different cells in our transformation procedure, the selection criteria for each component needed to be unique. In addition, we selected helper plasmids which had been known to work with the intended recipient cell.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/4a/PCPP33_tri-p_table.png" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Table 1</b> Components and selection criteria used in conjugation with the broad-host vector PCPP33 <br />
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f2/PRL1383A_tri-p_table.png" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Table 2</b> Components and selection criteria used in conjugation with the broad-host vector PRL1383A <br />
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<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Results<br />
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<p class="class">Testing the ligation of pRL1383a and BBa_I20260 using PCR and restriction digests showed that the insert was not present in the vector, and the conversion to BioBrick format ultimately unsuccessful. The procedure as described above was repeated multiple times without success. Tri-parental conjugation of unmodified pRL 1383a was inconclusive in all target organisms.</p><br />
<br />
<p class="class">In an effort to troubleshoot this vector, several different approaches were taken. First, the ligation was repeated with varying concentrations of insert (10X, 2X) in an attempt to account for the impact of the large vector size on the ligation reaction. These ligations yielded similar results to reactions done at calculated concentrations. A Blunt-end ligation using a Klenow fragment was also performed. This was repeated, both attempts without success. The BBa_I20260 PCR product with BamHI/HindIII ends was ligated into another vector in an attempt to test the insert’s ability to be cut with the restriction enzymes. This ligation did not indicate the presence of the insert, suggesting that the problem lies with the vector or primers. The primers were tested and found viable on another insert, with similar testing of restriction enzymes to show functionality. The primers and enzymes were operating as intended, but new enzymes were ordered for more experimental certainty. The insert was then digested only with HindIII, and left in a ligation reaction. The outcome of this ligation was not of the desired length. This was repeated, and the same result obtained. While there is some suggestion that the BioBrick insert may not be functioning, the ambiguous results of tri-parental mating with unmodified pRL1383a suggests that the vector may be damaged or misunderstood.</p><br />
<br />
<p class="class">Testing the ligation of PCPP33 and BBa_I20260 also proved unsuccessful. Restriction digests using BioBrick standard pieces failed to yield an insert. Tri-parental mating of this vector proved successful in all organisms that we tested. All organisms yielded colonies on tetracycline plates, suggesting presence of the plasmid. Further testing by plasmid extraction and gel analysis will be done to conclusively determine presence of the plasmid. <br />
</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/7/77/R_spaeroides_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/1/1e/P_putida_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/d/da/Synechocystis_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 4</b> Results of the tri-parental mating between pCPP33 and R. <i>sphaeroides</I>, P. <i>putida</i>, and Synechocystis sp., respectively. Each plate is shown alongside a negative control <br />
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Experiments: Secretion<br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20><br />
Methods for Constructing BioBrick Parts<br />
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<p class="class"><br />
One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length. <br />
<br />
<p class="class"><br />
Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.</p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Signal Peptides:</font></b><br><br />
<p class="class"><br />
To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.</p><br />
<p class="class"><br />
The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Phasin:</font></b><br><br />
<p class="class"><br />
The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis (LINK TO PROTOCOLS PAGE) (CTGCAG CTTCAG). Sequencing confirmed that this site was successfully removed. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>GFP:</font></b><br><br />
<p class="class"><br />
Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported. Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator. </p><br />
<br />
<p class="class"><br />
A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure X shows GFP- Top10 <i>E. coli</i> colonies (left) and unfused GFP+ Top10 <i>E. coli</i> colonies (right). This figure shows that the GFP construct is functional and easily detectable.</p><br><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/93/GFPglowingUSU.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 2.</b> Plate with GFP- cells (right) next to plate with GFP+ cells(left)<br />
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<br><b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Bioplastic Production:</font></b><br><br />
<p class="class">A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. <i>E. coli</i> harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure X. The observed peaks at 1.24 ppm, 2.54 ppm, and 5.2 ppm correspond with those observed in standard polyhydroxyalkanaote samples.</p><br />
<Br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/4/43/NMRusu.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Proton NMR spectra for PHB production in recombinant <i>E. coli</i><br />
</div><br />
<br><br />
<p class="class">To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>SDS-PAGE Analysis</font></b><br><br />
<p class="class">Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged (see Figure X). Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure X. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/d/d1/GFP_gel.png"" align = "middle" height="400" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing a strong band corresponding to GFP<br />
</div><br />
<br><br />
<p class="class">The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection, as shown in Figure X. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/3/3e/PHB_gel.png"" align = "middle" height="250" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing the presence of phasin protein in supernatant samples (third well from left)<br> next to supernatant from an <i>E. coli</i> sample without a phasin-producing construct.<br />
</div><br />
<Br><br />
<br />
<p class="class">Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Figure X shows the apparatus used to transfer proteins onto PVDF paper. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies. </p><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br /><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Future Work<br />
</font></b></i> <hr><br />
<ul class=circle><br />
<li>Resolve problems with conversion of broad-host range vectors to BioBrick compatible formats.</li><br />
<li>Further test the broad-host vectors to verify their functionality in additional organisms.</li><br />
<li>Further test different GFP and phasin constructs and determine additional ways to monitor phasin production, such as by mass spectrometry or transmission electron microscopy.</li><br />
<li>Design additional signal peptides that are functional with organisms like Synechocystis PCC6803, P. putida, and R. sphaeroides, and that can be expressed in BioBrick-compatible broad-host range vectors.</li><br />
<li>Optimize and expand both systems to include different protein products and organisms.</li><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>JUDGING</font></span><br />
<a href="#bronze">Bronze</a><br /><br />
<a href="#silver">Silver</a><br /><br />
<a href="#gold">Gold</a><br /><br />
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<b><i>BioBricks without Borders:</b></i></font><br />
<p><font face="Helvetica, Arial, San Serif" color =green>Investigating a multi-host BioBrick vector and secretion of cellular products</font></p><HR><br />
<p> <font size="2.5" face=" Tahoma, Helvetica, Arial" color =#000000><b>The requirements to earn a Bronze Medal are:</b><br><br />
n fulfillment of the requirements for the Gold Medal, the 2009 Utah State iGEM did the following:<br />
<br />
Bronze Medal Requirements<br />
<br />
* Completed the registration requirements and Project Summary form.<br />
* Prepared and will present a poster and talk at the 2009 Jamboree.<br />
* Entered all necessary information detailing 62 BioBricks into the Registry of Standard Parts.<br />
* Designed parts in conformity with accepted BioBrick standards.<br />
* DNA for 49 BioBricks entered in the Registry were sent in to iGEM headquarters. Each of these parts was confirmed with DNA sequencing.<br />
<br />
Silver Medal Requirements<br />
<br />
* Demonstrated that several submitted BioBricks work as expected.<br />
o The composite lac promoter/RBS/GFP/terminator (BBa_K2208045). This composite was demonstrated by the presence of GFP visualized on a UV transilluminator. The GFP protein was also visualized using SDS polyacrylamide gel electrophoresis. See pictures on Wiki. <br />
o New GFP reporter (BBa_K208000). This reporter, with an excitation/emission of 395/509, was shown to be functional using the novel composite construct BBa_K2208045 (lac promoter/RBS/GFP/terminator). Picture on Wiki under part BBa_K2208045. <br />
o The composite lac promoter/RBS (BBa_K208010). This was demonstrated using the composite construct BBa_K2208045. Picture on Wiki under part BBa_K2208045. This composite part is extremely useful because it alleviates the need to work with extremely small ribosomal BioBrick components. <br />
o Lac promoter/RBS/Gene III/Phasin/terminator (BBa_K208038). This was demonstrated through the detection of phasin proteins isolated from supernatant samples using SDS polyacrylamide gel electrophoresis. Picture on Wiki. <br />
o Phasin gene (BBa_K208001). A protein of the correct size was detected in SDS polyacrylamide gel electrophoresis from cells having this construct that was not detected in the control samples. <br />
o Gene III secretion tag (BBa_K208002). The phasin protein from part BBa_K208038 was detected outside the cell, thus demonstrating the functionality of this Gene III secretion tag.<br />
* Characterized the GFP reporter (BBa_K208000) (1) showing GFP expression over time and (2) showing sensitivity of IPTG induction.<br />
<br />
Gold Medal Requirements<br />
<br />
* Improved upon the existing set of GFP BioBrick parts by constructing a GFP with a larger Stokes shift (395/509nm), making for easier downsteam analysis. The bright fluorescence can very easily be seen on a standard UV transilluminator. As GFP is one of the most commonly used reporters, the introduction of this new and improved BioBrick part should be of great benefit to the iGEM community. <br />
* Our initial plans for our 2009 team was in part to continue the 2008 Hawaii team’s project, “Cyanobacteria Toolkit,” by making the pRL1383a vector an effective broad-host vector. When this vector proved ineffective, we made efforts to troubleshoot. We had much correspondence with the advisors of the Hawaii team, including a conference call mid-summer. Since most of their parts were not included in the 2009 iGEM distribution, we had them send many of the parts they used. After numerous approaches and attempts to convert pRL1383a into BioBrick format, we finally decided that the vector was either mischaracterized or altered in some unknown way. <br />
* As thoroughly explained in our Wiki, one of the primary goals of our project has been to expand the BioBrick world to organisms other than E. coli. We have documented the many benefits that would come from such an achievement. Though the pRL1383a vector efforts were unsuccessful, we did successfully express the vector pCPP33 in E. coli, Pseudomonas putida, Synechocystis pcc 6803, and Rhodobacter sphaeroides. This vector now has only to be put in BioBrick format. <br />
* In reference to our ethics section (https://2009.igem.org/Team:Utah_State/ETHICS), our team has taken specific measures to follow our suggested proposals. In the education of our team, we discussed the potential benefits of a standard secretion system but also discussed the potential of our designed secretion pathways to be used in a malevolent manner. As a team, we acknowledge the importance of high moral accountability and commitment to safety and security. Additionally, in an effort to foster the sharing of information in our community, upon completion of the jamboree we will submit an article to be released in our school newspaper and in the College of Engineering website. This winter we are also hosting a lecture by Drew Endy addressing synthetic biology which will be open to the public. We hope to act as ambassadors to foster support and excitement in our own community.<br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
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<a href="#references">References</a><br />
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Broad-Host Range Vectors<br />
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<br><br />
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Advantages for Using a Broad Host Range Vector<br />
</font></b></i> <br />
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<p class="class">A multi-host vector allows for genetic manipulation to occur in one organism, and the ultimate application of the vector to be served in another. Genetic manipulation is ideally done in <i>E. coli</i>, due to its fast growth, ease of use, and availability of transformable cells. However, it does not always represent the best choice for production of recombinant proteins or other compounds, and thus it is ideal to be able to transfer genetic information into other organisms once manipulation and testing of the construct is complete.</p><br />
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<p class="class">Most broad host range vectors are naturally occurring or a derivative of a natural vector. They tend to be large, around 10 kbp, although some commercial versions have been optimized to a much shorter length (http://www.bio101.com/functional-analysis/pBBR122.html). They can be self-transmissible (presence of <i>tra</i> genes) and mobilizable (mob genes), but desirable vectors are both mobilizable and non-transmissible (Haller, & Dichristina, 2002). This allows for more control over conjugation in the laboratory through use of a helper plasmid (Haller, & Dichristina, 2002). A helper plasmid is a conjugative plasmid, that is it contains both transmission and mobilization genes. While a broad-host range plasmid can be conjugated into another organism, its copy number will remain undetectably low unless a fully functioning helper plasmid is present (Haller, & Dichristina, 2002).. If a helper plasmid shares the same origin of transfer (oriT), mob genes are no longer necessary (Snyder and Champness 2007). Due to this property, the mob genes of commercial plasmids are often removed, thereby resulting in vectors that are significantly shorter than their natural counterparts (Snyder and Champness 2007). Use of a helper plasmid becomes necessary if the self-transmission genes are not present to achieve any detectable degree of replication in the recipient organism (Haller, & Dichristina, 2002).</p><br />
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Genetic Characteristics of Broad-Host Range Vectors<br />
</font></b></i><br />
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<p class="class">Broad host range vectors are a class of mobilizable plasmids, that is they lack the complete tra-genes necessary for conjugation but can still transfer and replicate at high copy number in the presence of a conjugative plasmid. Mobilizable vectors still contain some of the genes necessary for transfer. The mob genes code proteins that aid the vector in transferring from one organism to another. One protein produced in the region, nickase-helicase, nics the DNA at the origin of transfer (oriT). As the envelopes of the two cells meet, the mobility proteins synthesize a new strand of DNA from the plasmid parent strand as it enters the recipient cell. A new strand is also synthesized in the donor cell simultaneously. In this way, the plasmid is transferred from one cell to another (Porter, 2002).</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/Mechanism_of_Bacerial_Conjugation.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 1.</b> Mechanism for bacterial conjugation<br />
</div><br />
<br><br />
<p class="class">The multi-host vector pRL1383a was used in this study. It is derived from RSF1010, a naturally occurring broad host range vector found in <i>E. coli</i>. RSF 1010 has been completely sequenced (Scholz 1989). It is designed for use in Cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Two versions of this vector were tested: one containing mob A/B/C genes with an origin of transfer (Figure 2), and one utilizing an RP4 origin of transfer (matching the origin of transfer in the RP4 helper plasmid. This eliminates the need for mobilization genes when used with this helper vector). In addition, the vector has resistance cassettes for both Streptomycin and Spectinomycin (Wolk 2007).</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/SecretionTeam:Utah State/Secretion2009-10-22T03:17:36Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. <i>coli</i> MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. <i>coli</i> transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. <i>coli</i> or photoautotrophic PHA-producers R. <i>sphaeroides</i> and <i>Synechocystis</i> PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure 1 depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 1.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant <i>E. coli</i> do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure 2 depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. <i>coli</i> K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure 3. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative E. <i>coli</i> (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure 3 illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 3.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure 4 shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 4.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of E. <i>coli</i> (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in E. <i>coli</i> harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in E. <i>coli</i> (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 5 depicts the GFP barrel structure.</p><br />
<br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="200" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 5.</b> The GFP Barrel Structure<br />
</div><br />
<br />
<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. <i>coli</i>, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/Broad-HostVectorsTeam:Utah State/Broad-HostVectors2009-10-22T03:14:58Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Broad-Host Range Vectors<br />
</font></b></i> <hr><br />
<br />
<br><br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Advantages for Using a Broad Host Range Vector<br />
</font></b></i> <br />
<br><br />
<p class="class">A multi-host vector allows for genetic manipulation to occur in one organism, and the ultimate application of the vector to be served in another. Genetic manipulation is ideally done in <i>E. coli</i>, due to its fast growth, ease of use, and availability of transformable cells. However, it does not always represent the best choice for production of recombinant proteins or other compounds, and thus it is ideal to be able to transfer genetic information into other organisms once manipulation and testing of the construct is complete.</p><br />
<br />
<p class="class">Most broad host range vectors are naturally occurring or a derivative of a natural vector. They tend to be large, around 10 kbp, although some commercial versions have been optimized to a much shorter length (http://www.bio101.com/functional-analysis/pBBR122.html). They can be self-transmissible (presence of <i>tra</i> genes) and mobilizable (mob genes), but desirable vectors are both mobilizable and non-transmissible (Haller, & Dichristina, 2002). This allows for more control over conjugation in the laboratory through use of a helper plasmid (Haller, & Dichristina, 2002). A helper plasmid is a conjugative plasmid, that is it contains both transmission and mobilization genes. While a broad-host range plasmid can be conjugated into another organism, its copy number will remain undetectably low unless a fully functioning helper plasmid is present (Haller, & Dichristina, 2002).. If a helper plasmid shares the same origin of transfer (oriT), mob genes are no longer necessary (Snyder and Champness 2007). Due to this property, the mob genes of commercial plasmids are often removed, thereby resulting in vectors that are significantly shorter than their natural counterparts (Snyder and Champness 2007). Use of a helper plasmid becomes necessary if the self-transmission genes are not present to achieve any detectable degree of replication in the recipient organism (Haller, & Dichristina, 2002).</p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#000033><br />
Genetic Characteristics of Broad-Host Range Vectors<br />
</font></b></i><br />
<br />
<p class="class">Broad host range vectors are a class of mobilizable plasmids, that is they lack the complete tra-genes necessary for conjugation but can still transfer and replicate at high copy number in the presence of a conjugative plasmid. Mobilizable vectors still contain some of the genes necessary for transfer. The mob genes code proteins that aid the vector in transferring from one organism to another. One protein produced in the region, nickase-helicase, nics the DNA at the origin of transfer (oriT). As the envelopes of the two cells meet, the mobility proteins synthesize a new strand of DNA from the plasmid parent strand as it enters the recipient cell. A new strand is also synthesized in the donor cell simultaneously. In this way, the plasmid is transferred from one cell to another (Porter, 2002).</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/Mechanism_of_Bacerial_Conjugation.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 1.</b> Mechanism for bacterial conjugation<br />
</div><br />
<br><br />
<p class="class">The multi-host vector pRL1383a was used in this study. It is derived from RSF1010, a naturally occurring broad host range vector found in <i>E. coli</i>. RSF 1010 has been completely sequenced (Scholz 1989). It is designed for use in Cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Two versions of this vector were tested: one containing mob A/B/C genes with an origin of transfer (Figure 2), and one utilizing an RP4 origin of transfer (matching the origin of transfer in the RP4 helper plasmid. This eliminates the need for mobilization genes when used with this helper vector). In addition, the vector has resistance cassettes for both Streptomycin and Spectinomycin (Wolk 2007).</p><br />
<br />
<br />
<br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Experimental Section: Approach for BioBrick Compatibility<br />
</font></b></i><br />
<br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#000033><br />
Converting Broad-Host Vectors into a BioBrick-Compatible Format<br />
</font></b></i><br />
<p class="class">Two Broad-host range vectors were used in this study; pRL1383a and PCPP33. To convert these vectors into BioBrick-compatible format, the four standard BioBrick sites EcoRI, XbaI, SpeI, and PstI needed to be inserted into the multiple cloning site. For pRL1383a, common BioBrick primers VR and VF2 were also included to allow the use of PCR in amplifying the BioBrick parts.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/8/82/PRL1383A_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2</b> Plasmid map of pRL1383a <br />
</div><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/PCPP33_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 3</b> Plasmid map of pCPP33 <br />
</div><br />
<br><br />
<p class="class">Apart from being shown effective in the Synechosystis PCC 6803 (Marraccini 1993), pRL1383a is an ideal candidate for use as a BioBrick-compatible broad-host range vector because the BioBrick restriction sites are absent within the vector sequence. To convert pRL1383a into a BioBrick format, the existing multiple cloning site, which is flanked by a BamHI site and a HindIII site, was utilized. First, modified primers were synthesized from BioBrick primers VR and VF2. These primers were modified by adding extra nucleotides to insert the desired restriction enzyme sites into the PCR product. A BamHI site was added to 5’ end of the forward primer (VF2) and a HindIII site was added to the 5’ end of the reverse primer (VR). These primers were used to amplify an existing, tested BioBrick part by PCR. For this purpose, we selected BBa_I20260 because it does not contain BamHI or HindIII sites, and successful ligation is readily testable as it is a GFP -producing construct. The addition of IPTG is typically necessary to induce GFP production in this particular device. However, when using Top10 <i>E. coli</i> cells it is produced continuously because these cells lack a lac repressor (insert invitrogen link). After cutting the vector at the multiple cloning site using BamHI and HindIII, the BioBrick insert obtained by PCR with modified ends was ligated into the backbone. The vector was then transformed using Top10 One Shot® chemically competent <i>E. coli</i> and tested for successful insertion using PCR and restriction digests.</p><br />
<br />
<p class="class">Another broad host range vector, pCPP33, previously shown effective in Pseudomonas Putida,was standardized using similar methods. While the complete sequence of this plasmid is not available, it was shown that there are no BioBrick restriction sites outside the multiple cloning site (Figure 3). The multiple cloning site of this vector is flanked by EcoRI and HindIII. This allowed the PCR product of BBa_I20260 to again be used by cutting with HindIII and EcoRI restriction enzymes. Restriction digests and gel analysis were used to test for the insert.</p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#000033><br />
Broad Host Conjugation<br />
</font></b></i><br />
<br />
<p class="class">In order to transfer a vector of interest using conjugation, the <i>tra</i> gene (contained in what we will refer to as a transfer plasmid, or helper plasmid) must be expressed in order to initiate the conjugation process. This plasmid codes for genes which, when expressed, form pili on the cell surface, which in turn initiate conjugation (Heinemann 1989). This plasmid may be present in one of three different procedures:</p><br />
<ul><br />
</li><li><b>Hfr strain</b> – The <i>tra</i> operon is many times contained in an episome, which can incorporate itself into the cell genome. These resultant Hfr strains will often begin the transfer of their own DNA, both plasmid and genomic. Due to the transfer of the genomic DNA, these strains are referred to as high frequency recombinant (Hfr) strains.<br />
<br />
</li><li><b>Biparental (normal) Conjugation</b> – Cells containing the <i>tra</i> genes, often labeled as F-positive (F+) due to the F-plasmid, a well-known transfer plasmid, can express the transfer genes necessary for conjugation to occur. When a vector of interest and a transfer plasmid are of different incompatibility groups, they may both be transformed into the same cell, and conjugation may take place between the F+ donor cell and the recipient cell<br />
<br />
</li><li><b>Triparental Mating</b> – In the case where the transfer plasmid and the vector of interest are of the same incompatibility group, the two plasmids may not stably coexist (Heinemann 1989). In this case, two separate cells containing the transfer gene (the helper cell) and the vector (the donor cell) must be used in conjugation. The helper cell will assist the donor cell in the transfer of its mobilizable plasmid to the recipient cell. This method circumvents some of the barriers that may prevent the transfer of plasmids.<br />
</ul><br />
<br />
<br />
<p class="class">For our project, we chose to use the triparental mating procedure for the transmission of our vector. While not being the most efficient method, it circumvents possible barriers and intermediate steps.</p><br />
<br />
<p class="class">Because of the use of three different cells in our transformation procedure, the selection criteria for each component needed to be unique. In addition, we selected helper plasmids which had been known to work with the intended recipient cell.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/4a/PCPP33_tri-p_table.png" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Table 1</b> Components and selection criteria used in conjugation with the broad-host vector PCPP33 <br />
</div><br />
<br><br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f2/PRL1383A_tri-p_table.png" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Table 2</b> Components and selection criteria used in conjugation with the broad-host vector PRL1383A <br />
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<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Results<br />
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<br />
<p class="class">Testing the ligation of pRL1383a and BBa_I20260 using PCR and restriction digests showed that the insert was not present in the vector, and the conversion to BioBrick format ultimately unsuccessful. The procedure as described above was repeated multiple times without success. Tri-parental conjugation of unmodified pRL 1383a was inconclusive in all target organisms.</p><br />
<br />
<p class="class">In an effort to troubleshoot this vector, several different approaches were taken. First, the ligation was repeated with varying concentrations of insert (10X, 2X) in an attempt to account for the impact of the large vector size on the ligation reaction. These ligations yielded similar results to reactions done at calculated concentrations. A Blunt-end ligation using a Klenow fragment was also performed. This was repeated, both attempts without success. The BBa_I20260 PCR product with BamHI/HindIII ends was ligated into another vector in an attempt to test the insert’s ability to be cut with the restriction enzymes. This ligation did not indicate the presence of the insert, suggesting that the problem lies with the vector or primers. The primers were tested and found viable on another insert, with similar testing of restriction enzymes to show functionality. The primers and enzymes were operating as intended, but new enzymes were ordered for more experimental certainty. The insert was then digested only with HindIII, and left in a ligation reaction. The outcome of this ligation was not of the desired length. This was repeated, and the same result obtained. While there is some suggestion that the BioBrick insert may not be functioning, the ambiguous results of tri-parental mating with unmodified pRL1383a suggests that the vector may be damaged or misunderstood.</p><br />
<br />
<p class="class">Testing the ligation of PCPP33 and BBa_I20260 also proved unsuccessful. Restriction digests using BioBrick standard pieces failed to yield an insert. Tri-parental mating of this vector proved successful in all organisms that we tested. All organisms yielded colonies on tetracycline plates, suggesting presence of the plasmid. Further testing by plasmid extraction and gel analysis will be done to conclusively determine presence of the plasmid. <br />
</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/7/77/R_spaeroides_PCPP33.JPG" align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/1/1e/P_putida_PCPP33.JPG" align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/d/da/Synechocystis_PCPP33.JPG" align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 4</b> Results of the tri-parental mating between pCPP33 and <i>R. sphaeroides</I>, <i>P. putida</i>, and Synechocystis sp., respectively. Each plate is shown alongside a negative control <br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/TeamTeam:Utah State/Team2009-10-22T03:13:46Z<p>Liblint: </p>
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<a href="https://2009.igem.org/Team:Utah_State"><br />
<img alt="USU iGem" src="https://static.igem.org/mediawiki/2009/7/71/USUlogo.jpg"/> </a><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>ABOUT US</font></span><br />
<a href="#team">The Team</a><br /><br />
<a href="#advisors">Advisors</a><br /><br />
<a href="#faculty">Faculty Support</a><br /><br />
<a href="#USU">Utah State University</a><br /><br />
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<b><i>Team USU</i></b></font><br />
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<p> <font size="2.5" face=" Tahoma, Helvetica, Arial" color =#000000><br />
This is Utah State University's second year participating in the iGEM competition. Our team has grown in the past year and includes high school, undergraduate, and graduate students, along with outstanding faculty advisors. Though there have been many challenges throughout this project, participation in the iGEM competition has been a great learning experience for all involved on the team. Below, you can meet each member of our team and find more information about Utah State University and Logan, Utah. </font></p><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ee/Teamusu.png"" align = "middle" height="400" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<img src="https://static.igem.org/mediawiki/2009/f/fc/Garrett_Hinton_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Garrett Hinton"/> <br />
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<b><br />
GARRETT HINTON<br />
</b></font><p class="margin"><br />
Garrett Hinton is a Senior at Sky View High School in Smithfield, UT. He began his research experience at USU in 2006 as he was starting his freshman year in high school. He was a member of the USU iGEM team in 2008, and is excited to be involved again this year. For this project, Garrett performed various lab procedures, including DNA purification, gel electrophoresis, restriction enzyme digestions, ligations, and more. Additionally, he helped out in the lab by making medias and monitoring experiments. Outside of the lab, Garrett likes basketball and ping pong.<br />
</p><br/><br/><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/b/b3/Jody_Jerez_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Jody Jerez"/> <br />
<font size="2" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b><br />
JODY JEREZ<br />
</b></font><br />
<p class="margin">Jody Jerez is a senior at InTech Collegiate High School in North Logan, Utah. She has been interested in biological engineering and decided to participate in iGEM to learn more about it. This is her first year participating in iGEM and she has learned a lot and enjoys what she was able to do for the team. Jody contributed by doing general laboratory procedures in the lab, as well as adding content to the wiki. Other than learning more about biological engineering, Jody enjoys hiking, dancing, and rock climbing.<br />
</p><br /><br /><br /><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/3/39/Jeff_Karren_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Jeff Karren"/> <br />
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<br />
<b><br />
JEFF KARREN<br />
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<p class="margin"><br />
Jeff is also a senior at InTech Collegiate High School. He first learned about iGEM and synthetic biology when USU's Biological Engineering gave a presentation at his school. Although Jeff helped primarily with the team wiki, especially the protocol page, he did help with some work in the lab as well, particularly in the initial stages of the project. He has enjoyed learning about synthetic biology, and is excited for the Jamboree. Jeff is on his high school robotics team, plays the pipe organ, hikes, and swing dances. <br />
</p><br /><br /><br /><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/5/56/Tyrell_Rupp_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Tyrell Rupp"/> <br />
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<b><br />
TYREL RUPP<br />
</b></font><br />
<p class="margin">Tyrel is a senior at a Sky View High School near USU. In the spring of 2009 he was exposed to synthetic biological engineering through an internship class. Ty spent the summer working for the Synthetic Biomanufacturing Center, which lead to his participation in iGEM. Over the past few months, he has learned a lot about synthetic biology and the research process. Among other things, Ty enjoys mountain biking and snowboarding in the mountain ranges above USU. For iGEM, Ty has helped carry out basic laboratory procedures and contributed some to the team’s wiki design. <br />
</p><br /><br /><br /><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/c/c2/Hyun-jin_Kim_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Tyrell Rupp"/> <br />
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<b><br />
HYUN-JIN KIM<br />
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<p class="margin">Hyun-Jin is a freshman at Intech Collegiate High School. He started at the lab from summer and is now very proficient in most laboratory procedures used in the Synthetic Biomanufacturing Lab. In example, he makes media, isolates plasmid DNA using the CTAB method, and carries out transformations of <i> E. coli</i>. He is from Gwangju, South Korea and is excited to be involved with the iGEM 2009 project. He enjoys football, Frisbee and basketball.<br />
</p><br /><br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/7/79/Sean_Bedingfield_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Sean Bedingfield"/> <br />
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<b><br />
SEAN BEDINGFIELD<br />
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<p class="margin">Sean Bedingfield is a Utah State Research Fellow in his freshman year. His personal research is focused on effective expression of the LacOperon gene for the production of particular proteins. Sean is an avid hiker, pheasant hunter, and cook. Sean’s contributions to the project include assisting with tri-parental mating, researching<br />
secretion systems of different bacteria, transforming competent <i>E. coli</i>, and getting the team T-shirts made. <br />
</p><br /><br /><br /><br /><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/1/1d/Cole_Peterson_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Cole Peterson"/> <br />
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<b><br />
COLE PETERSON<br />
</b></font><br />
<p class="margin">Cole is a sophomore in Biological Engineering, and this is his first year participating in iGEM. He spent the summer enthusiastically working on USU’s iGEM project. When not in school or in the lab he likes running, cycling, hiking, rock climbing and skiing. Cole spent the majority of his time in the lab modifying and testing multiple broad-host range vectors, as well as performing general laboratory tasks and procedures.<br />
</p><br /><br /><br /><br /><br /><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/3/37/Alex_Hatch_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Alex Hatch"/> <br />
<font size="2" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b><br />
ALEX HATCH<br />
</b></font><br />
<p class="margin">Alex Hatch is a junior in Biological Engineering at Utah State University. His personal research has dealt with microbial diversity in TCE bioremediation and currently algal diversity in wastewater treatment and biofuel production. He is participating with iGEM for the first time this year and has enjoyed the opportunity to be introduced to this emerging field. He is pursuing a career in medicine and plans to attend medical school upon graduation. He loves to spend time with his young family, wife Laura and son Graham. Together they like to spend time outside, read together, and build forts out of furniture and blankets. Alex has helped in general laboratory activities and was responsible for addressing human practices in synthetic biology on the Wiki.</p><br />
<br /><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/e/ea/Rachel_Jackson_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Rachel Porter"/> <br />
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<b><br />
RACHEL PORTER<br />
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<p class="margin">Rachel is in her third year at Utah State University majoring in Biological Engineering. She is interested in genetic and biomedical engineering research and would like to pursue a masters degree in biomedical engineering. She enjoys participating in iGEM because it is interesting and helps her apply the concepts she has learned in classes. She hopes to work in a research laboratory after graduation. Originally from Salt Lake City, Utah, her hobbies include playing volleyball, mountain biking, hiking, camping, and almost anything outdoors. <br />
<br /><br /><br /></p><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/b/b7/Trent_Mortensen_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Trent Mortensen"/> <br />
<font size="2" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b><br />
TRENT MORTENSEN<br />
</b></font><br />
<p class="margin">Trent is a finishing senior in Biological Engineering at Utah State University. His personal research is focused on the antimycobacterial properties of the St. John’s Wort herb. Trent participated in the 2008 Utah State iGEM project and is participating once again because of the excellent experience he had last year. He is looking into a career in the biomedical field in the area of disease and injury research and treatment and feels that Synthetic Biology has enormous potential for advancing this field. Trent enjoys hiking, fishing, and snowmobiling in the nearby mountains. He also enjoys playing basketball, racquetball, and ultimate. For this year’s project, Trent aided in laboratory work, team coordination, gathering materials for the Wiki, and preparation of the presentation materials. </p><br />
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<img src=" https://static.igem.org/mediawiki/2009/3/32/Brad_Henrie_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Brad Henrie" /> <br />
<font size="2" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b><br />
BRAD HENRIE<br />
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<p class="margin">Brad is a graduate student in the Biological and Irrigation Engineering at Utah State University. His personal research is centered around analyzing an unknown microbial community, and characterizing it's genetic phylogeny. Brad is participating in the iGEM competition for the first time, and has enjoyed being a part of the group. He has a passion for caving, and tries to go as often as the team would let him. Other than that, he enjoys rock climbing, camping, the occasional computer game, and is excited for the duck hunting season. Brad was primarily responsible for culturing the cyanobacteria, and conducting the tri-parental mating procedure, and generally helped out around the lab.<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7f/Libbie_Linton_cropped.jpg" align = "right" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Libbie Linton" /> <br />
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LIBBIE LINTON<br />
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<p class="margin">Libbie is a Masters student in Biological Engineering. Like a few others on this year's team, Libbie was also a member of the 2008 iGEM team. She is glad to be back for a second year, this time as an advisor. Her Masters' work is in investigating different ways to improve PHA production economics. She is hoping to complete her thesis within the next few months, followed by finding employment in bioprocess/biochemical engineering. In her spare time, Libbie is a musician. She also enjoys beating fellow iGEM teammate Trent Mortensen in racquetball, as well as playing with her 6 month old puppy, Rooster. For this years' iGEM team, Libbie advised on topics related to bioplastic production, secretion systems and mechanisms, and general laboratory procedures. She was also involved in coordinating efforts with the wiki. <br />
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<img src="https://static.igem.org/mediawiki/2008/7/7f/JH.jpg" align = "right" height="158" style="padding:.5px; border-style:solid; border-color:#999" alt="Junling Huo" /> <br />
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JUNLING HUO<br />
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<p class="margin">Junling Huo is a PhD student in Biological Engineering at USU. His dissertation research is focused on design a gene expression system for Rhodobacter sphaeroides, which is a photosynthetic bacteria. This gene expression system will include BioBrick compatible promoters, Ribosome Binding Sites (RBS), and terminators. The planned promoters and RBS will have different activities. Terminators will be either bidirectional or unidirectional. He hopes to complete his dissertation in the next year or so, and continuing doing research. He was also a member of 2008 USU iGEM team, and is happy to serve as an advisor again this year.<br />
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Our faculty advisors are Dr. Ronald C. Sims, Dean H. Scott Hinton, and Dr. Charles D. Miller. We would like to thank them for all of their help and guidance throughout the project. To view their professional biographies, please refer to our <a href="http://www.bie.usu.edu/"><b><font color =#009900>department website</font></b></a>:<br />
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<b><i>Utah State University</i></b></font><br />
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Located in <a href="http://www.loganutah.org/"><b><font color=#009900>Logan, Utah</font></b></a>, about 80 miles north of Salt Lake City, Utah State University sits just outside the mouth of Logan Canyon. The city, founded in 1859, has a population of approximately 47,000 and boasts a rich heritage. Whether on campus, historic Main Street, down in the “island”, or up the canyon, there is always something to do. <br />
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Originally founded in 1888 as the Agricultural College of Utah, the school officially became a university in 1957. As the state's land-and space-grant university, USU conducts world-class research in many disciplines and has several projects in conjunction with the Department of Defense and NASA. USU’s research program is second in age only to MIT in the United States. USU's <a href="http://www.sdl.usu.edu/index-noflash.html"><b><font color =#009900>Space Dynamics Laboratory</font></b></a> has put more experiments into space than any university in the world and is ranked first in the United States for funding for aerospace research. <br />
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<p class="class">The <a href="http://www.engineering.usu.edu"><b><font color=#009900>engineering</font></b></a> program at Utah State is known for its excellence, with a 96% first-time pass rate on the national engineering exam compared to a national average of 55%. Beyond the Logan campus, Utah State's Extension programs extend academic resources and support throughout the entire state of Utah, having extension locations in each of Utah's 29 counties.</p><br />
<p class="class"><a href="http://www.bie.usu.edu/"><b><font color =#009900>Biological and Irrigation Engineering</font></b></a> is a relatively small department at Utah State, with about 30 faculty and 120 undergraduate and graduate students. This great student-to-teacher ratio helps students succeed in their classes. The Biological Engineering Program teaches students to manipulate biological systems for useful purposes, understand scientific literature, and to work well and communicate effectively with others, both in the field of Biological Engineering and out. In the first years of the program, students learn the basics of biology, chemistry, physics, and mathematics. This knowledge base is then broadened by a study of liberal arts and humanities including literature, philosophy, political science, art, and music classes. Students finally delve into technical engineering courses, many chosen personally by students to apply to their particular areas of interest. These courses develop practical problem-solving abilities, increasing sensitivity to the economic, social, and legal dimensions of technical problems. Students leave the program well-qualified for their careers, with an understanding of the importance of social and professional ethics and responsibility to accompany their technical learning. After graduation, students will apply their knowledge to a wide range of careers, from genetic engineering to design of prosthetic devices for amputees.</p><br />
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<b><i>Welcome!</b></i></font><br />
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<p class="class"><br />
The Utah State University team is excited to be participating in the 2009 iGEM competition. We invite you to explore our site and learn all about our project! And please contact us if you would like more information about any aspect of our project.</p><br />
<p class = "class"> The aim of the Utah State University iGEM project is to develop improved production and harvesting methods of proteins and other products in multiple organisms using the standardized BioBrick system. The name of our project, BioBricks without Borders, characterizes and ties together the two main focuses of our research:</p></br><br />
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<li>Investigating broad host-range vectors for production of compounds in organisms other than <i>E. coli</i> (like <i>Synechocystis</i> PCC6803, <i>Rhodobacter sphaeroides</i>, and <i>Pseduomonas putida</i>)<br />
<li>Developing a library of fusion-compatible BioBrick parts for targeting compounds for secretion</li><br />
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<p class="class">The USU iGEM team has successfully constructed 49 BioBrick parts and devices. Of these, we have had time to demonstrate the functionality of 8 BioBricks, and had 41 of them sequenced. Check out our <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Utah_State/"><font color=#009900>BioBrick Registry</font></b></a> page to see all of these parts and to find more detailed information about them. The following figures shows all of the potential different combinations of pieces used to make composite devices for this project.<br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>BIOBRICKS</font></span><br />
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<b><i>BioBricks</b></i></font><br />
<p class="class">The USU iGEM team has successfully constructed 49 BioBrick parts and devices. Of these, we have had time to demonstrate the functionality of 8 BioBricks, and had 41 of them sequenced. Check out our <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Utah_State/"><font color=#009900>BioBrick Registry</font></b></a> page to see all of these parts and to find more detailed information about them. The following figures shows all of the potential different combinations of pieces used to make composite devices for this project.<br />
</p><br />
<div align="center"> <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Utah_State"><img src="https://static.igem.org/mediawiki/2009/c/ca/Slide5USU.jpg " align = "middle" height="300" style="float:left; alt="SBC"> </a> </div><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/ModelingTeam:Utah State/Modeling2009-10-22T03:06:21Z<p>Liblint: </p>
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<a href="#parameters">Parameters</a><br /><br />
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<b><i>Modeling Secretion Mechanisms</b></i></font><br />
<HR><br />
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<p class = "class"> <br />
There are currently few guidelines for selecting which signal peptide (which determine the secretion pathway taken) should be used for a given recombinant protein (Choi, 2004). The construction of models that evaluate protein secretion may provide a useful framework for studying and attempting to predict factors that affect secretion efficiency. Models of the Sec and Tat translocation pathways were made using MATLAB’s Simbiology toolbox. They were evaluated using the embedded ordinary differential equation solver in the software. Assumptions were made for both models due to lack of time scale information for individual steps for these translocation mechanisms. However, both provide a flexible framework that can become more detailed as additional information is found in literature or discovered in the laboratory. Within both models, a protein is first generated and then carried out of the cytoplasm to the periplasm. Only protein species were tracked, as species involved with making the protein are currently not as useful to monitor.<br />
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Model Parameters<br />
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<p class = "class"> <br />
In both models, parameters for the manufacture of protein were the same and found using averaged values for <i>E. coli</i>. The average length of an mRNA for <i>E. coli</i> is 1100 nucleotides and an <i>E. coli</i> cell transcribes at an average rate of 70 nucleotides per second. A simple rate calculation determines that a strand of mRNA is made approximately every 15.7 seconds. This value was then used to yield a first order reaction rate of (1/15.7) 1/second. The translation rate was found in a similar manner. Given an average protein size in <i>E. coli</i> as 360 amino acids and the average translation rate is 40 amino acids per second, the first order translation rate constant is (1/9) 1/s (Institute for Biomolecular Design, 2008). To initiate the model, the concentration of the gene was set at 1, which was locked at that value over the course of the simulation. The median half life for mRNA was found to be 3.7 minutes, after which it will degrade (Milo, n. d.). For the Tat-dependent mechanism, the protein is fully folded prior to translocation (Mergulhão, 2005). For this folding time, a place holder value of 10 minutes was used as an approximate order of magnitude, although the value can easily be changed depending on parameters of the protein in question. Degradation of protein was modeled as the average time it takes the cell to reproduce, which is given as half an hour (Institute for Biomolecular Design, 2008).<br />
</P><br />
<p class = "class"> <br />
In addition to the discussed values regarding the manufacture of protein, parameters for the Sec and Tat pathway are required. A literature review yielded no specific rate constants or time parameters for the individual steps involved with either process. However, one article stated that the Sec and Tat pathways take a few seconds and a few minutes, respectively, to translocate protein to the periplasmic space (Mergulhão, 2005). Accordingly, the Sec and Tat pathways were modeled to require 3 seconds and 3 minutes for protein translocation, respectively. First order rate constants were determined by taking the inverse of these values in seconds. Images of the model diagrams are seen below. The different pathways after the formation of protein can be activated and deactivated as more detailed information comes available about the translocation and secretion processes. The Sec secretion pathway also includes the SRP secretion pathway as a possible option. The constructed models for the Sec and Tat pathways are given as Figures 1 and 2, respectively.<br />
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<b>Figure 1.</b> Sec Pathway Model Diagram<br />
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<b>Figure 2.</b> Tat Pathway Model Diagram<br />
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Simulations<br />
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<p class = "class">Simulations for the Sec pathways were run for 5400 seconds (1.5 hours), which allowed the number of periplasmic to reach approximately steady state. The simulation for the Sec pathway secretion model is shown below as Figure 3. <br />
Protein generated by the <i>E. coli</i> cell reaches steady state quickly, as it is both produced and translocated in to the periplasm rapidly. The periplasmic protein reaches approximately 90 proteins upon achieving steady state. The long settling time and high number of periplasmic protein are a result of the long protein degradation time.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/a3/ModifiedGraph1.jpg" align = "middle" height="400" style="padding:.5px; border-style:solid; border-color:#999" alt="Figure 3"> </div><br />
<br><br />
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<b>Figure 3.</b> Simulation of protein production and translocation by the Sec pathway<br />
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<br />
<p class= "class">Simulations for the Tat pathway were run for 3600 seconds (1 hour). The shorter length of simulation relative to the Sec model is used despite having significantly longer protein translocation time because the time-consuming folding step limits the reaction. The results of the ordinary differential equation simulation are shown below in Figure 4. The amount of periplasmic protein is lower than cytoplasmic and folded protein values upon reaching steady state. This is due to the rate-limiting folding step, which also leads to an expected high concentration of folded protein as well.<br />
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<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/d/d2/ModifiedGraph2.jpg" align = "middle" height="400" style="padding:.5px; border-style:solid; border-color:#999" alt="Figure 3"> </div><br />
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<b>Figure 3.</b> Simulation of protein production and translocation by the Sec pathway<br />
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<br />
<p class = "class"> The accuracy of models, such as those constructed, will increase as more knowledge about the time step parameters for each of the mechanisms is determined. These improvements will allow for a better comparison of the two secretion methods, which can lead to better signal peptide selection (and corresponding pathway selection) for the protein in question. Future work could also include incorporating promoter strength into the model to affect transcription rates to provide a more accurate depiction of what is happening inside the cell. Better correlation between these parameters and the model will result in a more useful aid in when studying protein secretion. <br />
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References<br />
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<ul class = "circle"><br />
<li>Institute for Biomolecular Design (2008, August 1) CyberCell Database. Retrieved October 2009. http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi<br />
<li>Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using <i>Escherichia coli</i>. Appl Microbiol Biotechnol 64:625-635</li><br />
<li>Mergulhão FJM, Summers DK, Montier GA (2005) Recombinant protein secretion in <i>Escherichia coli</i>. Biotechnol Adv 23:177-202</li><br />
<li>Milo R (n.d.) BioNumbers: The Database of Useful Biological Numbers. Retrieved October 2009. http://bionumbers.hms.harvard.edu/default.aspx</li><br />
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<a href="#parameters">Parameters</a><br /><br />
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<b><i>Modeling Secretion Mechanisms</b></i></font><br />
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There are currently few guidelines for selecting which signal peptide (which determine the secretion pathway taken) should be used for a given recombinant protein (Choi, 2004). The construction of models that evaluate protein secretion may provide a useful framework for studying and attempting to predict factors that affect secretion efficiency. Models of the Sec and Tat translocation pathways were made using MATLAB’s Simbiology toolbox. They were evaluated using the embedded ordinary differential equation solver in the software. Assumptions were made for both models due to lack of time scale information for individual steps for these translocation mechanisms. However, both provide a flexible framework that can become more detailed as additional information is found in literature or discovered in the laboratory. Within both models, a protein is first generated and then carried out of the cytoplasm to the periplasm. Only protein species were tracked, as species involved with making the protein are currently not as useful to monitor.<br />
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Model Parameters<br />
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In both models, parameters for the manufacture of protein were the same and found using averaged values for <i>E. coli</i>. The average length of an mRNA for <i>E. coli</i> is 1100 nucleotides and an <i>E. coli</i> cell transcribes at an average rate of 70 nucleotides per second. A simple rate calculation determines that a strand of mRNA is made approximately every 15.7 seconds. This value was then used to yield a first order reaction rate of (1/15.7) 1/second. The translation rate was found in a similar manner. Given an average protein size in <i>E. coli</i> as 360 amino acids and the average translation rate is 40 amino acids per second, the first order translation rate constant is (1/9) 1/s (Institute for Biomolecular Design, 2008). To initiate the model, the concentration of the gene was set at 1, which was locked at that value over the course of the simulation. The median half life for mRNA was found to be 3.7 minutes, after which it will degrade (Milo, n. d.). For the Tat-dependent mechanism, the protein is fully folded prior to translocation (Mergulhão, 2005). For this folding time, a place holder value of 10 minutes was used as an approximate order of magnitude, although the value can easily be changed depending on parameters of the protein in question. Degradation of protein was modeled as the average time it takes the cell to reproduce, which is given as half an hour (Institute for Biomolecular Design, 2008).<br />
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In addition to the discussed values regarding the manufacture of protein, parameters for the Sec and Tat pathway are required. A literature review yielded no specific rate constants or time parameters for the individual steps involved with either process. However, one article stated that the Sec and Tat pathways take a few seconds and a few minutes, respectively, to translocate protein to the periplasmic space (Mergulhão, 2005). Accordingly, the Sec and Tat pathways were modeled to require 3 seconds and 3 minutes for protein translocation, respectively. First order rate constants were determined by taking the inverse of these values in seconds. Images of the model diagrams are seen below. The different pathways after the formation of protein can be activated and deactivated as more detailed information comes available about the translocation and secretion processes. The Sec secretion pathway also includes the SRP secretion pathway as a possible option. The constructed models for the Sec and Tat pathways are given as Figures 1 and 2, respectively.<br />
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<b>Figure 1.</b> Sec Pathway Model Diagram<br />
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<b>Figure 2.</b> Tat Pathway Model Diagram<br />
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Simulations<br />
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<p class = "class">Simulations for the Sec pathways were run for 5400 seconds (1.5 hours), which allowed the number of periplasmic to reach approximately steady state. The simulation for the Sec pathway secretion model is shown below as Figure 3. <br />
Protein generated by the <i>E. coli</i> cell reaches steady state quickly, as it is both produced and translocated in to the periplasm rapidly. The periplasmic protein reaches approximately 90 proteins upon achieving steady state. The long settling time and high number of periplasmic protein are a result of the long protein degradation time.</p><br />
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<b>Figure 3.</b> Simulation of protein production and translocation by the Sec pathway<br />
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<p class= "class">Simulations for the Tat pathway were run for 3600 seconds (1 hour). The shorter length of simulation relative to the Sec model is used despite having significantly longer protein translocation time because the time-consuming folding step limits the reaction. The results of the ordinary differential equation simulation are shown below in Figure 4. The amount of periplasmic protein is lower than cytoplasmic and folded protein values upon reaching steady state. This is due to the rate-limiting folding step, which also leads to an expected high concentration of folded protein as well.<br />
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<b>Figure 3.</b> Simulation of protein production and translocation by the Sec pathway<br />
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<p class = "class"> The accuracy of models, such as those constructed, will increase as more knowledge about the time step parameters for each of the mechanisms is determined. These improvements will allow for a better comparison of the two secretion methods, which can lead to better signal peptide selection (and corresponding pathway selection) for the protein in question. Future work could also include incorporating promoter strength into the model to affect transcription rates to provide a more accurate depiction of what is happening inside the cell. Better correlation between these parameters and the model will result in a more useful aid in when studying protein secretion. <br />
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References<br />
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<li>Institute for Biomolecular Design (2008, August 1) CyberCell Database. Retrieved October 2009. http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi<br />
<li>Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using <i>Escherichia coli</i>. Appl Microbiol Biotechnol 64:625-635</li><br />
<li>Mergulhão FJM, Summers DK, Montier GA (2005) Recombinant protein secretion in <i>Escherichia coli</i>. Biotechnol Adv 23:177-202</li><br />
<li>Milo R (n.d.) BioNumbers: The Database of Useful Biological Numbers. Retrieved October 2009. http://bionumbers.hms.harvard.edu/default.aspx</li><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
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References<br />
</font></b></i> <hr><br />
<ul class=circle><br />
<li>Akita M, Sasaki S, Matsuyama S, Mizushima S (1990) J Biol Chem 265:8164-8169</li><br />
<li>Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450-472</li><br />
<li>Barrett CML, Ray N, Thomas JD, Robinson C, Bolhuis A (2003) Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli. Biochem BIophys Res Comm 304:279-284</li><br />
<li>Berks BC (1996) Mol Microbiol 22:393-404</li><br />
<li>Berks BC, Sargent F, Palmer T (2000) The Tat protein export pathway. Mol Microbiol 35:260-274</li><br />
<li>Binet,R, Letoffe S, Ghigo JM, Delepelaire P, Wandersman C. Protein secretion by Gram-negative bacterial ABC exporters-a review. Gene 1997;192:7-11</li><br />
<li>Blight MA, Holland IB (1994) Heterologous protein secretion and the versatile <i>Escherichia coli</i> haemolysin translocator. Trends Biotechnol 12:450-455</li><br />
<li>Cao TB, Saier MH (2003) The general protein secretory pathway:phlyogenetic analyses leading to evolutionary conclusions. Biochimica et Biophysica Acta 1609:115-125</li><br />
<li>Chalfie M, Tu Y, Euskirchen G, Ward W, and Prasher D. (1994) Green fluorescent protein as a marker for gene expression. Science 263:802-805 </li><br />
<li>Choi J (1999) Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation. Appl Microbiol Biot 51:13-21</li><br />
<li>Choi J, Lee SY (1997) Process analysis and economic evaluation for poly-β-hydroxybutyrate production by fermentation. Bioprocess Eng 17:335-342</li><br />
<li>Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using <i>Escherichia coli</i> Appl Microbiol Biotechnol 64:625-635</li><br />
<li>Cianciotto NP (2005) Type II Secretion: A Protein Secretion System for all Seasons. Trends in Microbiology 13:581-588</li><br />
<li>Crameri A, Whitehorn E, Tate E, Stemmer W. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotech 14:315-19. </li><br />
<li>Cristobal S, de Gier JW, Nielsen H, von Heijne G (1999) Competition between Sec- and Tat-dependent protein translocation in <i>Escherichia coli</i> EMBO J 18:2982-2990 </li><br />
<li>Desveaux M, Parham N, Scott-Tucker A, Henderson IR (2004) The general secretory pathway: a general misnomer. Trends Micro 12:306-309</li><br />
<li>Doi Y (1990) Microbial Polyester. VCH, New York<br />
Fernandez LA, de Lorenzo V (2001) Fomration of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway. Mol Microbiol 40:332-346</li><br />
<li>Gentschev I, Goebel W (2003) Type I protein secretion systems in gram-negative bacteria: <i>Escherichia coli</i> alpha-hemolysin secretion. In: Oudega B, editor. Kluwer Academic Publishers, pp.121-139</li><br />
<li>Hearn MT, Acosta D (2001) Applications of novel affinity cassette methods: Use of peptide fusion handles for the purification of recombinant proteins. J Mol Recognit 14:323-369</li><br />
<li>Haller C.A., & Dichristina, T.J. (2002). Genetic approaches in bacteria with no natural genetic systems. New York, NY: Wiley-Liss, Inc. </li><br />
<li>Hess J, Gentschev I, Goebel W, Jarchau T (1990) Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins. Mol Gen Genet 224:201-208</li><br />
<li>Invitrogen Corporation (2009) Cloning of A-tailed PCR fragments using conventional ligase method. Retrieved October 2009. https://commerce.invitrogen.com</li><br />
<lI>Jung IL, Phyo KH, Kim KC, Park HK, Kim IG (2005) Spontaneous liberation of intracellular polyhydroxybutyrate granules in <i>Escherichia coli</i>. Res Microbiol 156:865-873</li><br />
<li>Kaderbhai MA, Davey HM, Kaderbhai NN (1997) A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge. Protein Science 13:2458-2469</li><br />
<Li>Koster M, Bitter W, Tommassen J (2000) Protein secretion mechanisms in Gram-negative bacteria. Int J Med Microbiol 290:325-331</li><br />
<li>Lavallie ER, McCoy JM (1995) Gene fusion expression systems in <i>Escherichia coli</i>.Current Biology Ltd 501-506</li><br />
<li>Lee SY (1996) Bacterial Polyhydroxyalkanoates. Biotechnol Bioeng. 49:1-14<br />
<li>Luirink J, Oudega B Protein targeting to the inner membrane. In: Oudega B, editor. Kluwer Academic Publishers, pp 1-21</li><br />
<Li>Madison LL, Huisman GW (1999) Metabolic engineering of poly (3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol R 63:21-53</li><br />
<li>Maehara A, Ueda S, Nakano H, Yamane T (1999) Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of <i>Paracoccus denitrificans</i>. J Bacteriol 191:2914-2921</li><br />
<li>Mergulhão, FJM, Summers DK, Monteiro (2005) Recombinant protein secretion in <i>Escherichia coli</i> Biotechnol Adv 23:177-202</li><br />
<li>Molhoj M (2004) Leader sequences are not signal peptides. Nat Biotech 22:1502</li><br />
<li>Palmer T, Berks BC (2004) The Tat protein export pathway In: Oudega B, editor. Kluwer Academic Publishers, pp 51-64</li><br />
<li>Phillips I, Silver P (2006) A new biobrick assembly strategy designed for facile protein engineering. </li><br />
<li>Porter RD (2002) Modern microbial genetics, 2nd ed. New York, NY: Wiley-Liss Inc. </li><br />
<lI>Prieto MA (2007) From Oil to Bioplastics, a Dream Come True? J Bacteriol 189(2):289-290</li><br />
<li>Pugsley AP (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57:50-108</li><br />
<li>Resch S, Gruber K, Wanner G, Slater S, Dennis D, and W Lubitz (1998) Aqueous release and purification of poly(β-hydroxybutyrate) from Escherichia coli. J Biotechnol 65:173-182. </li><br />
<li>Sabirova JS, Ferror M, Lunsdorf H, Wray V, Kalscheuer R, Steinbuchel A, Timmis KN, Golyshin PN (2006) Mutation in a “<i>tesB</i>-Like” Hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxalkanoates by <i>Alcanivorax borkumensis</i> SK2. J Bacteriol 188:8452-8459</li><br />
<li>Sandkvist M (2001) Biology of type II secretion. Mol Microbiol 40:271-83</li><br />
<li>Sandkvist M, Bagdasarian M (1996) Secretion of recombinant proteins by Gram-negative bacteria</li><br />
<li>Santini C, Bernadac A, Zhang M, Chanal A, Ize B, Blan co C, Wu L (2001) Translocation of jellyfish green fluorescent protein via the Tat system of <i> Escherichia coli</i> and change of its periplasmic localization in response to osmotic up-shock. J Biol Chem 276:8159-8164</li><br />
<li>Sapriel G, Wandersman C, Delepelaire P (2003) The SecB chaperone is bifnctional in <i>Serratia marcescens</i>: SecB is involved in the Sec pathway and required for HasA secretion by the ABC transporter. J Bacteriol 185:80-88</li><br />
<li>Schatz PJ, Beckwith J (1990) Genetic analysis of protein export in <i>Escherichia coli</i> Annu Rev Genet 24:215-48</li><br />
<li>Scholtz (1989) Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene 75:217-288</li><br />
<li>Shimomura O, Johnson F, and Saiga Y. (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, <i>Aequorea</i>. J.Cell Comp Physiol 59:223-39 </li><br />
<li>Shokri A, Saden AM, Larsson G (2003) Cell and process design for targeting of recombinant protein into the culture medium of <i>Escherichia coli</i>. Appl Microbiol Biotechnol 60:654-664</li><br />
<li>Snyder, Champness (2007) Molecular Genetics of Bacteria, 3rd ed., ASM Press</li><br />
<li>Steinbüchel A, Füchtenbusch B (1998) Bacterial and other biological systems for polyester production. Trend Biotechnol 16:419-27</li><br />
<li>Suriyanmongkol P, Weselake R, Narine S, Moloney M, Shah S (2007) Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants – A review. Biotechnol Adv. 25:148-175</li><br />
<li>Thomas JD, Daniel RA, errington J, Robinson C (2001) Export of Active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in <i> Escherichia coli</i>. Mol Microbiol 39:47-53</li><br />
<li>Valent QA, Kendall DA, High S, Kusters R, Oudega B, Luirink J (1995) EMBO J 14:5494-5505</li><br />
<li>van der Does C, Noewen N, Driessen AJM (2004) The Sec translocase. In: Oudega B, editor. Kluwer Academic Publishers, pp:23-49</li><br />
<li>Veenendaal AKL, van der Does C, Driessen AJM (2004) The protein-conducting channel SecYEG. Biochimica et Biophysica Act 1694:81-95</li><br />
<li>Verlinden RAJ, Hill DJ, Kenward MA, Williams CD, Radecka I (2007) Bacterial synthesis of biodegradable polyhydroxyalkanoates. J Appl Microbiol 102:1437-1449</li><br />
<li>Von Heijne G (1986) Net N-C charge imbalance may be important for signal sequence function in bacteria. J Mol Biol 192:287-290</li><br />
<li>Von Heijne G (1996) Principles of membrane protein assembly and structure. Prog Biophys Mol Bio 66:113-139</li><br />
<li>Wolk CP, Fan Q, Zhou R, Huang G, Lechno-Yossef S, Kuritz T, Wojciuch E. (2007) Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120. Arch. Microbiol. 188:551-563</li><br />
<li>York GM, Junker BH, Stubbe J, Sinskey AJ (2001) Accumulation of the PhaP Phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells. J Bacteriol 183:4217-4226</li><br />
<li>York GM, Stubbe J, Sinkskey AJ (2002) New insight into the role of the PhaP phasin of <i>Ralstonia eutropha</i> in promoting synthesis of polyhydroxybutyrate. J Bacteriol 183:2394-2397</li><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/NotebookTeam:Utah State/Notebook2009-10-22T02:11:34Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"<font size = 4>NOTEBOOK</font></span><br />
<a href="#meeting">Meeting Notes </a><br /><br />
<a href="#notebook">Lab Notebook</a><br /><br />
<a href="#protocols">Protocols</a><br/><br />
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Official Meetings <br />
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<p class="header">May 12</p><br />
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<p class = "class">Introduction to team members and to iGEM. Reviewed last year’s competition and last year’s team contribution. Introduction also to the 2009 iGEM home page and our team wiki. </p><br />
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<p class="header">June 23</p><br />
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<p class = "class">After researching previous projects and brainstorming since our last meeting, we discussed the possibility of continuing the project that University of Hawaii initiated last year. We spoke with an advisor from their team and agreed that we could continue the project. Our team agreed that it would be a good project foundation.</p> <br />
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<p class="header">June 30</p><br />
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<p class = "class">Small workshop on programming a wiki. Further collaboration with team Hawaii has taken place and we are waiting for DNA constructs with which they worked. </p><br />
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<p class="header">August 20</p><br />
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<p class = "class">Discussed progress made with broad-host vector construction. The broad-host vector constructs that we have been working with from Team Hawaii and from the iGEM parts catalog do not appear to be functioning. After PCR, attempted ligations, enzymatic digestions and electrophoretic gel observations, we’ve decided to move on and try to modify another known broad-host vector to be compatible with the BioBrick format. </p><br />
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<p class="header">September 3</p><br />
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<p class = "class">Team discussion of PHAs, phasin, silver-fusion, and secretory pathways. Further discussion of attempted broad-host vector modifications. </p><br />
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<p class="header">September 10</p><br />
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<p class = "class">Reviewed judging criteria and reviewed our standing with the broad-host vector and the secretion pathways. Discussed which tracks would be most applicable to our project. Discussed titles for our project. Finalization of team roster and travel information. </p><br />
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<p class="header">September 17</p><br><br />
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<p class = "class">Made final decisions for our intended track, chose a final project title, and gave a final review of our abstract. Discussed our wiki progress. </p><br />
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<p class="header">September 24</p><br><br />
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<p class = "class">Took team picture. Presentation of different team logo options and team shirt design options. Flash animation presentation to be used potentially in wiki and presentation. <br />
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<p class="header">September 29</p><br><br />
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<p class = "class">Team meeting with internet programming advisor. Discussed final formatting options for wiki. </p><br />
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<p class="header">October 1</p><br><br />
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<p class = "class">Instruction given on tri-parental mating. Discussed selective plates and media for tri-parental mating. </p><br />
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<p class="header">October 6</p><br><br />
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<p class = "class">Instruction given on Western Blot procedure and function. Discussed modified GFP construct. Final team logo was presented.</p><br />
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<p class="header">October 8</p><br><br />
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<br />
<p class = "class">Met shortly and separated to work on different assignments for wiki. </p><br />
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<p class="header">October 13</p><br><br />
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<p class = "class">Final T-shirt design presented to team. Reviewed completed parts and discussed broad-host vector. Presentation of completed secretion pathway models. </p><br />
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<p class="header">October 15</p><br><br />
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<p class = "class">Discussed portions of the wiki that needed to be completed before the weekend. Reviewed project components and iGEM basics in preparation for the jamboree. </p><br />
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<p class="header">October 20</p><br><br />
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<p class = "class">Final meeting before wiki closure. Discussed last minute assignments to ensure that the wiki is completed.</p><br />
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Laboratory Notebook<br />
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<p class="class"><br />
Members of our team each had individual lab notebooks. Rather than outline each procedure that was run by each individual person, we have instead decided to present our wiki lab notebook as a weekly update up the progress that was made. Many of the various procedure details and specifics are found below in the protocols section. </p><br />
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<b>Figure 1.</b> Some of our laboratory notebooks<br />
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Protocols<br />
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<p class="class"><br />
Bacterial Transformation <br />
<br />
Once the target DNA has been successfully ligated into the plasmid vector, the plasmid must be transferred into the host cell for replication and cloning. In order to do this, the bacterial cells must first be made “competent.” The term “competent” is to describe a cell state in which there exist gaps or openings in the cell wall which will allow the plasmid containing the target genes to enter into the cell. Several methods to make bacterial cells competent exist, such as the calcium chloride method and electroporation. Competent cells may also be purchased commercially. The team at USU has purchased competent cells for all experiments. The following is the method used by the USU team to insert the plasmids containing various biobricks into the cells. <br />
<br />
Method <br />
<br />
1. Ensure the necessary antibiotic agar plates have been prepared or begin their preparation now. Four plates per transformation will be necessary (two today, then two tomorrow for streaking). Also ensure that 10 ml liquid media is made up per transformation (also for tomorrow).<br />
<br />
2. If using Biobrick parts from iGEM distribution, use registry to identify appropriate will containing plasmid of interest and proceed to step 3, if using other DNA proceed to step 5.<br />
<br />
3. Add 10ul of sterile water to distribution well to dissolve DNA. Remove 10ul and place in 0.5ml bullet tube. Label tube with part number, use 2ul to transform and save the other 8ul in the BioBrick part box.<br />
<br />
4. Take competent cells from the -80˚C freezer and place on an ice bath.<br />
<br />
5. Add 2 μl of the DNA solution (or 4ul of ligation reaction) to the competent cells. Ensure the pipetting is done directly into the cell solution. Let cells incubate on ice for 30 minutes. Heat water bath to 42˚C.<br />
<br />
6. Heat shock cells in the 42˚C water bath for 30 seconds. Remove and place back in the ice bath for 2 minutes.<br />
<br />
7. In the hood, add 250 μl SOC media to each tube, bringing the total cell solution to 300 μl. Incubate at 37˚C for 1 hour.<br />
<br />
8. Add 200 μl of each transformed cell solution to the appropriate antibiotic plate. Use the Bunsen burner to create a “hockey stick” out of a glass pipette tip by holding over the flame until it bends. Allow to cool. Spread cell solution uniformly over the agar plate using the “hockey stick,” then before discarding, spread residual solution on the “stick” over a second plate to get more a more sparse colony distribution. <br />
<br />
9. Parafilm all plates and place in 37˚C incubator 12-14 hours, or overnight if that is not possible. <br />
<br />
<br />
Streak Plates and Liquid Cultures from Transformed Colonies <br />
<br />
After bacterial cells have been transformed, successfully transformed cells must be selected. Because 100% of the cells do not receive the desired plasmid and target gene, it is essential to select for cells that do have the target genes. The USU team uses antibiotic resistance to select for successful transformations. To do this, an antibiotic resistance gene is also added to the plasmid vector that contains the target genes. By doing so, it is possible to know that a cell was successfully transformed based on its ability to grow on an agar plate with antibiotics added. Because the cell is able to grow, the antibiotic resistance gene must be present as well as the target gene. From the agar plates containing the antibiotics, a colony is picked and transferred into a liquid culture for further analysis. The following is the method used by USU to clone the DNA and select for the successful transformation of various BioBricks in E.coli. <br />
<br />
Method<br />
<br />
1. Prepare two 15 ml tubes per transformation, each with 5 ml media containing the appropriate antibiotic. <br />
<br />
2. Use a pipette tip to extract half of each colony and inoculate one agar plate per colony. Using a pipette with a tip, extract the other half of each colony and inoculate one liquid media tube per colony. Label all tubes and plates and place in the 37˚C incubator until the next morning. <br />
<br />
<br />
<br />
Plasmid DNA Isolation <br />
<br />
Following successful bacterial cloning and isolation, it is important to verify that the target gene is in the cell and that the resultant plasmid is correct. To do this, it is a common practice to sequence the plasmid DNA. To obtain enough DNA for sequencing, the bacterial clones are grown in a liquid culture. The cells are harvested by centrifugation and then prepared for DNA plasmid extraction. DNA plasmid extraction can be done several ways, and the overall purpose is to lyse the cells and separate the plasmid DNA from all other cellular proteins, DNA, and debris. The following is the method used by the USU team to isolate plasmid DNA containing the various biobricks. <br />
<br />
Method <br />
<br />
1. Prepare two water baths, one boiling and the other 68C. <br />
<br />
2. Centrifuge bactrerial cultures (3 to 5 ml) at 3K RPM for 20 min. Discard supernatant. <br />
<br />
3. Resuspend cell pellet in 200 μl of STET buffer. Transfer to 1.5 ml tubes.<br />
<br />
4. Add 10 μl of lysozyme (50 mg/ml) and incubate at room temperature for 5 min. <br />
<br />
5. Boil for 45 sec and centrifuge for 20 min at 13K RPM (or until pellet gets tight).<br />
<br />
6. Use a pipette tip or toothpick to remove the pellet.<br />
<br />
7. Add 5 μl RNase A (10 mg/ml) to supernatant and incubate at 68C for 10 minutes.<br />
<br />
8. Add 10 μl of 5% CTAB and incubate at room temperature for 3 min.<br />
<br />
9. Centrifuge for 5 min at 13K RPM, discard supernatant, and resuspend in 300 μl of 1.2 M NaCl by vortexing.<br />
<br />
10. Add 750 μl of ethanol and centrifuge for 5 min at 13K RPM.<br />
<br />
11. Discard supernatant, rinse pellet (which cannot be seen) in 80% ethanol, and let tubes dry upside down with caps open.<br />
<br />
12. Resuspend pellet in either sterile water or TE buffer. <br />
<br />
<br />
Restriction Enzyme Digestion and Electrophoresis <br />
<br />
Restriction enzyme digestion is the process by which an insert DNA sequence is separated from the rest of the DNA molecule. Specific knowledge of the DNA insert is needed to determine which enzyme and conditions to use during the digestion reaction. Once the DNA sequence is known and the correct enzymes have been selected, the DNA may be digested. Listed below is the procedure used by USU to digest the plasmid DNA. After enzyme digestion, electrophoresis is used to separate the plasmid from the insert. A gel is prepared and the respective reaction mixes are loaded into the gel. Using a DNA ladder, and knowing the size of the insert, the corresponding band can be seen and cut out of the gel. The insert may then be removed and isolated from the gel, thus yielding the desired DNA. The DNA from this may then be used in PCR reactions, sequencing, ligations for further experimentation, etc. Listed below are example protocols used by the USU team for a restriction enzyme digestion and subsequent agarose gel electrophoresis. <br />
<br />
<br />
Method <br />
<br />
1. Resuspend DNA in 20 to 40 μl water, vortex, and do a brief centrifuge to get solution to the bottom of the tube. <br />
<br />
2. Add components to the digestion solution in the following order: DNA (23 μl), 10X restriction enzyme buffer (3 μl), Xba1 (2 μl), and Pst1 (2 μl). The volume and restriction enzymes can be varied, but it should be ensured that the total volume is 10X the amount of RE buffer. Tap tubes periodically and allow to digest at appropriate temperature while preparing electrophoresis gel. <br />
<br />
3. Prepare electrophoresis gel by adding 2 g agarose to 200 ml TAE (1% solution). This is best done in an Erlenmeyer flask of adequate volume as swirling will need to be done. Place in the microwave and microwave on high for 20 seconds at a time, pulling it out and swirling until solution is homogeneous again, then repeating (BE CAREFUL to watch the solution closely when swirling – it superheats and can boil over and cause severe burns). Continue until solution is seen boiling in the microwave then gently swirl again. <br />
<br />
4. Add 20 μl ethidium bromide to solution and swirl until dissolved evenly. <br />
<br />
5. Add 6 μl of 6X loading dye to each tube of digested DNA solution. <br />
<br />
6. Prepare the electrophoresis unit by orienting the basin sideways with rubber gaskets firmly against the side. Place desired well template in the basin. <br />
<br />
7. When the agarose solution is cool enough to comfortably touch the flask, pour into the basin until the solution is about ¾ of the way to the top of the well template. <br />
<br />
8. When the gel is solidified (should look somewhat cloudy), remove the well template and change basin orientation to have the wells closest to the negative pole (as the DNA will flow towards the positive pole). Pour 1X TAE buffer into both sides of the electrophoresis unit until it just covers the gel and fills the wells.<br />
<br />
9. By inserting the pipette tip below the TAE liquid and into the well, add 10 μl of DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the digested DNA solutions to the wells by adding about 2 μl less than the total volume in the tubes to prevent air bubbles in the wells.<br />
<br />
10. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 70 V (this can be as high as 100 V if time is an issue), and press the start button. Separation should take two to three hours. The yellow dye shows the location of the smaller nucleotide lengths and the blue dye shows the location of the larger nucleotide lengths. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. The basin can then be placed back in the electrophoresis unit for further separation if desired. Take care to not have the power supply on without the lid to the unit in place. <br />
<br />
11. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking. Place the cone-shaped cover over the transilluminator and place the digital camera in the top hole for pictures. <br />
<br />
<br />
Media Preparation <br />
<br />
<br />
For all experimentation involving the need for bacterial biomass and experimentation, proper media is needed to grow the cells. The following is the media composition and methods used by USU to prepare the media. <br />
<br />
<br />
1. Add 5 g yeast extract, 10 g NaCl, 10 g Bacto Tryptone, and 15 g agar (if desired) to a 2 L Erlenmeyer flask and bring the volume up to 1 L with ddH20. Mix by swirling. Cover top with foil.<br />
<br />
2. Autoclave for 45 minutes (liquid setting, 0 minutes drying time). It will take an additional half hour for the autoclave to finish cooling then an additional 20 to 30 minutes until the media is cool enough to pour. <br />
<br />
<br />
1X TAE Preparation <br />
<br />
1. Add 40 ml 50X TAE solution to a 2 L flask and bring level up to 2 L with ddH20. <br />
<br />
<br />
Polymerase Chain Reaction (PCR) <br />
<br />
PCR is used to amplify a desired DNA sequence. The reaction is first set up by designing primers that will bind only to the desired regions of the DNA sequence. Once the primer and polymerase have been selected, the reaction parameters of time and temperature must be optimized. When the reaction works properly only the target DNA will be amplified into large quantities that may then be isolated and used for further experimentation. The following is the procedure used by USU for PCR reactions to amplify various biological parts. A useful set of primers are the universal BioBrick primers VF2 and VR that can be used to amplify almost any BioBrick part. <br />
<br />
Method<br />
<br />
1. Obtain the following reagents from the freezer: DNA template (cells or DNA), 10X Taq buffer (+KCl, -Mg/Cl2), MgCl2, 10 mM dNTP Mix, Taq polymerase (take out of freezer only immediately when needed and put back), and sterile distilled H2O. Place all reagents on ice. Also obtain PCR (either 0.2 or 0.5 ml) tubes.<br />
<br />
2. Add the following reagents to a tube (50 μl reaction) in the following volumes and order:<br />
<br />
• 32 μl sterile H2O,<br />
<br />
• 5 μl 10X buffer,<br />
<br />
• 2 μl dNTP Mix,<br />
<br />
• 6 μl MgCl2<br />
<br />
• 3 μl cells/DNA,<br />
<br />
• 0.25 μl Taq Polymerase<br />
<br />
• 1 μl primer 1<br />
<br />
• 1 μl primer 2 <br />
<br />
MgCl2 volume can be varied (lower to increase specificity – just ensure total volume is 50 ul with H2O). If many reactions are to be constructed, a master mix can be made up to cut down on time and pipette tip usage (if this is done, ensure primers are added to the appropriate reaction, i.e. perhaps not to the master mix). Tap or vortex tubes and take to the thermocyler. Place all reagents back in the -20˚C freezer.<br />
<br />
3. Choose thermocycler temperatures. The Eppendorf Mastercycler will cycle between three temperatures: typical temperatures are 94˚C for denaturing, 50-60˚C for primer annealing, and 72˚C for polymerase extending. Lowering the annealing temperature decreases DNA specificity; 55˚C is a good temperature to begin if no trials have been made with the sample.<br />
<br />
4. Turn on thermocycler with the switch in the back of the unit and open the lid. The placement of the tubes depends on the size of the tube (0.2 or 0.5 ml) and whether or not a temperature gradient is to be used.<br />
<br />
▪If no temperature gradient will be used, tubes can be placed anywhere on the unit in the appropriately-sized hole. Select “Files” and press enter. Select “Load” and then “Standard.” If cells will be used in the reaction, include a 1-minute lysing step at the beginning (step 1); this will be followed by a 1-minute DNA denaturing step (step 2). If purified DNA will be used, set step 1 to 1 second. Set an annealing temperature for step 3. Ensure the lid temperature is 105˚C and the extending temperature is 72˚C. Press exit. If prompted to save, save by pressing enter three times. Press exit to return to the main menu. Choose “Start” on the main menu and select “Standard.” The program should begin.<br />
<br />
▪If a temperature gradient is to be used, temperature will vary according to column. A 20˚C range is the maximum range that can be used (+/- 10˚C). The range is made by setting a temperature for the middle column and then setting a +/- range. To see what the temperatures will be if a gradient is used, select “OPTIONS” on the main menu, then select “Gradient.” Select the size tube that is being used by pressing “Sel,” then press enter. Choose a temperature for the center column, press enter, then select a +/- range and press enter. The column number along with the corresponding temperature is shown. Decide tube placement based on this information. Press exit twice to return on the main menu. Select “Files” then “Load,” then “Gradient.” If cells are being used, set the cell lysing step (step 1) to 1 minute (1:00); if purified DNA is being used, set this time to 1 second (0:01). Step 2 should be 94C, Step 4 should be 72˚C, and the lid temperature should be 105˚C. Go to step 3 and set an annealing temperature for the center column. Leave the next two lines as they are, and change the gradient setting (“G”) to the +/- the center temperature amount. Press exit. If prompted to save, press enter three times; if not prompted to save, press enter once. Press exit to get back to the main menu. To begin cycle, select “Start,” then select “Gradient.” The program should begin.<br />
<br />
5. The thermocycler is set to store the completed reaction tubes at 4˚C when finished. <br />
<br />
<br />
Ligation <br />
<br />
Ligation is the process by which the insert (target DNA gene) is inserted into a plasmid. Both the plasmid and insert have been digested and have the proper “sticky” or blunt ends which are compatible for joining the two DNA pieces together into one molecule. These two DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid. This newly formed plasmid may then be isolated using gel electrophoresis and then used for bacterial transformation or other experimentation. The following is the procedure used by USU to ligate together various biobrick parts. <br />
<br />
Method <br />
<br />
1. Obtain the following reagents, some of which are in the -20˚C freezer: DNA vector, DNA insert, 10X ligation buffer, T4 DNA ligase (take out only when needed, then return immediately to freezer), and sterile distilled water.<br />
<br />
2. Ideally, it is desirable to have the concentration of insert ends (or moles of insert) be two to three times the concentration of vector ends (or moles of vector), with a total DNA concentration of 50-400 ng/μl in the reaction. If determining the DNA concentration is not possible, place two to three times the volume of vector as the volume of insert in the reaction. As this is often the case, place the following reagents in a thin-walled PCR tube in the following volumes:<br />
<br />
• 10 μl insert DNA<br />
<br />
• 3 μl vector DNA<br />
<br />
• 2 μl 10X ligation buffer<br />
<br />
• 4 μl H2O<br />
<br />
• 1 μl T4 DNA ligase<br />
<br />
= 20 μl total<br />
<br />
This could also be done in different volumes depending on DNA concentration/total volume desired.<br />
<br />
3. Gently mix the tube, and place the tube in the PCR thermocyler, turn on the machine, select “Start,” from the main menu, select “22” and press “Start.” The thermocycler will keep the reaction at 22˚C.<br />
<br />
4. Incubate for 60 minutes. Heat-inactivate by placing tubes in 68C water bath for 10 minutes. Place in the freezer if storing for later use. <br />
<br />
<br />
<br />
<br />
<br />
<br />
Western Blot <br />
<br />
Western blotting is a procedure that allows for the identification of proteins using a specific antibody after protein separation on an SDS polyacrylamide gel. <br />
<br />
Method <br />
<br />
<ul class="circle"><br />
<li>Collect bacterial cells by centrifugation and lyse the cells using any of a variety of procedures.</li><br />
<li>Spin at 14,000 rpm (16,000 g) in a microfuge for 10 min at 4°C.</li><br />
<li>Transfer the supernatant to a new tube and discard the pellet.</li><br />
<li>Determine the protein concentration (Bradford assay, A280, or BCA)</li><br />
<li>Mix 20 µl of sample (20ug) with 10ul of 3x sample buffer.</li><br />
<li>Boil for 1 min, ool at RT for 5 min.</li><br />
<li>Flash spin to bring down condensation prior to loading gel.</li><br />
<li>Assemble pre-prepared polyacrylamide gel into gel running rig.</li><br />
<li>Load protein samples into individual wells.</li><br />
<li>Use 10 µl of Kaleidoscope standard.</li><br />
<li>Run at 35 mA (constant current) for approximately 2hrs.</li><br />
<li>Disassemble gel when done running.</li><br />
<li>Cut a piece of PVDF membrane (Millipore Immobion-P #IPVH 000 10).</li><br />
<li>Wet for about 10 min in methanol on a rocker at room temp.</li><br />
<li>Remove methanol and add 1x Blotting buffer until ready to use.</li><br />
<li>Assemble "sandwich" for Bio-Rad's Transblot.</li><br />
<li>Sponge - filter paper - gel - membrane - filter paper - sponge</li><br />
<li>Transfer for 1 hr at 100V at 4°C on a stir plate. Bigger proteins might take longer to transfer.</li><br />
<li>When finished, immerse membrane in blocking buffer (5% nonfat dry milk)and block overnight.</li><br />
<li>Incubate with primary antibody diluted in 0.5% blocking buffer for 60 min at room temp.</li><br />
<li>Wash 3 x 10 min with 0.05% Tween 20 in PBS.</li><br />
<li> Incubate with secondary antibody diluted in 0.5% blocking buffer for 45 min at room temp.</li><br />
<li>Wash 3 x 10 min with 0.05% Tween 20 in PBS.</li><br />
<li>Detect with TMB stabilized substrate for HRP. </li><br />
<br />
</ul><br />
Site-Directed Mutagenesis<br />
<br />
QuikChange II Site-Directed Mutagenesis Kit (Stratagene) <br />
<br />
<br />
1. Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence.<br />
<br />
2. Prepare the control reaction as indicated below:<br />
<br />
▪5 μl of 10× reaction buffer (see Preparation of Media and Reagents)<br />
<br />
▪2 μl (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/μl)<br />
<br />
▪1.25 μl (125 ng) of oligonucleotide control primer #1 [34-mer (100 ng/μl)]<br />
<br />
▪1.25 μl (125 ng) of oligonucleotide control primer #2 [34-mer (100 ng/μl)]<br />
<br />
▪1 μl of dNTP mix<br />
<br />
▪39.5 μl of double-distilled water (ddH2O) to a final volume of 50 μl<br />
<br />
Then add<br />
<br />
▪ 1 μl of PfuTurbo DNA polymerase (2.5 U/μl)<br />
<br />
3. Prepare the sample reaction(s) as indicated below: <br />
<br />
Note: Set up a series of sample reactions using various concentrations of dsDNA template ranging from 5 to 50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA template) while keeping the primer concentration constant. <br />
<br />
▪ 5 μl of 10× reaction buffer<br />
<br />
▪ X μl (5–50 ng) of dsDNA template<br />
<br />
▪ X μl (125 ng) of oligonucleotide primer #1<br />
<br />
▪ X μl (125 ng) of oligonucleotide primer #2<br />
<br />
▪ 1 μl of dNTP mix<br />
<br />
▪ ddH2O to a final volume of 50 μl<br />
<br />
Then add<br />
<br />
▪ 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) <br />
<br />
4. If the thermal cycler to be used does not have a hot-top assembly, overlay each reaction with ~30 μl of mineral oil.<br />
<br />
5. Cycle each reaction using the cycling parameters as outlined in Table I of the<br />
<br />
Stratagene QuikChange II Site-Directed Mutagenesis Kit manual. We used an annealing temperature of 55C for 1 min and an extension temperature of 68C for 5 min and 18 cycles.<br />
<br />
6. Following temperature cycling, place the reaction on ice for 2 minutes to cool the reaction to ≤37°C. If desired, amplification may be checked by electrophoresis of 10 μl of the product on a 1% agarose gel. A band may or may not be visualized at this stage. In either case proceed with Dpn I digestion and transformation. <br />
<br />
Dpn I Digestion of the Amplification Products<br />
<br />
1. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction below the mineral oil overlay using a small, pointed pipet tip.<br />
<br />
2. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37°C for 1 hour to digest the parental (i.e., the nonmutated) supercoiled dsDNA. <br />
<br />
Transform into XL1-Blue Supercompetent Cells and proceed as previously described. </p><br />
<br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
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<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
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Experimental Section: Approach for BioBrick Compatibility<br />
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Converting Broad-Host Vectors into a BioBrick-Compatible Format<br />
</font></b></i><br />
<p class="class">Two Broad-host range vectors were used in this study; pRL1383a and PCPP33. To convert these vectors into BioBrick-compatible format, the four standard BioBrick sites EcoRI, XbaI, SpeI, and PstI needed to be inserted into the multiple cloning site. For pRL1383a, common BioBrick primers VR and VF2 were also included to allow the use of PCR in amplifying the BioBrick parts.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/8/82/PRL1383A_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2</b> Plasmid map of pRL1383a <br />
</div><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/PCPP33_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 3</b> Plasmid map of pCPP33 <br />
</div><br />
<br><br />
<p class="class">Apart from being shown effective in the Synechosystis PCC 6803 (Marraccini 1993), pRL1383a is an ideal candidate for use as a BioBrick-compatible broad-host range vector because the BioBrick restriction sites are absent within the vector sequence. To convert pRL1383a into a BioBrick format, the existing multiple cloning site, which is flanked by a BamHI site and a HindIII site, was utilized. First, modified primers were synthesized from BioBrick primers VR and VF2. These primers were modified by adding extra nucleotides to insert the desired restriction enzyme sites into the PCR product. A BamHI site was added to 5’ end of the forward primer (VF2) and a HindIII site was added to the 5’ end of the reverse primer (VR). These primers were used to amplify an existing, tested BioBrick part by PCR. For this purpose, we selected BBa_I20260 because it does not contain BamHI or HindIII sites, and successful ligation is readily testable as it is a GFP -producing construct. The addition of IPTG is typically necessary to induce GFP production in this particular device. However, when using Top10 <i>E. coli</i> cells it is produced continuously because these cells lack a lac repressor (insert invitrogen link). After cutting the vector at the multiple cloning site using BamHI and HindIII, the BioBrick insert obtained by PCR with modified ends was ligated into the backbone. The vector was then transformed using Top10 One Shot® chemically competent <i>E. coli</i> and tested for successful insertion using PCR and restriction digests.</p><br />
<br />
<p class="class">Another broad host range vector, pCPP33, previously shown effective in Pseudomonas Putida,was standardized using similar methods. While the complete sequence of this plasmid is not available, it was shown that there are no BioBrick restriction sites outside the multiple cloning site (Figure 3). The multiple cloning site of this vector is flanked by EcoRI and HindIII. This allowed the PCR product of BBa_I20260 to again be used by cutting with HindIII and EcoRI restriction enzymes. Restriction digests and gel analysis were used to test for the insert.</p><br />
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Broad Host Conjuation<br />
</font></b></i><br />
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<p class="class">In order to transfer a vector of interest using conjugation, the <i>tra</i> gene (contained in what we will refer to as a transfer plasmid, or helper plasmid) must be expressed in order to initiate the conjugation process. This plasmid codes for genes which, when expressed, form pili on the cell surface, which in turn initiate conjugation (Heinemann 1989). This plasmid may be present in one of three different procedures:</p><br />
<ul><br />
</li><li><b>Hfr strain</b> – The <i>tra</i> operon is many times contained in an episome, which can incorporate itself into the cell genome. These resultant Hfr strains will often begin the transfer of their own DNA, both plasmid and genomic. Due to the transfer of the genomic DNA, these strains are referred to as high frequency recombinant (Hfr) strains.<br />
<br />
</li><li><b>Biparental (normal) Conjugation</b> – Cells containing the <i>tra</i> genes, often labeled as F-positive (F+) due to the F-plasmid, a well-known transfer plasmid, can express the transfer genes necessary for conjugation to occur. When a vector of interest and a transfer plasmid are of different incompatibility groups, they may both be transformed into the same cell, and conjugation may take place between the F+ donor cell and the recipient cell<br />
<br />
</li><li><b>Triparental Mating</b> – In the case where the transfer plasmid and the vector of interest are of the same incompatibility group, the two plasmids may not stably coexist (Heinemann 1989). In this case, two separate cells containing the transfer gene (the helper cell) and the vector (the donor cell) must be used in conjugation. The helper cell will assist the donor cell in the transfer of its mobilizable plasmid to the recipient cell. This method circumvents some of the barriers that may prevent the transfer of plasmids.<br />
</ul><br />
<br />
<br />
<p class="class">For our project, we chose to use the triparental mating procedure for the transmission of our vector. While not being the most efficient method, it circumvents possible barriers and intermediate steps.</p><br />
<br />
<p class="class">Because of the use of three different cells in our transformation procedure, the selection criteria for each component needed to be unique. In addition, we selected helper plasmids which had been known to work with the intended recipient cell.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/4a/PCPP33_tri-p_table.png" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Table 1</b> Components and selection criteria used in conjugation with the broad-host vector PCPP33 <br />
</div><br />
<br><br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f2/PRL1383A_tri-p_table.png" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Table 2</b> Components and selection criteria used in conjugation with the broad-host vector PRL1383A <br />
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<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Results<br />
</font></b></i><br />
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<br />
<p class="class">Testing the ligation of pRL1383a and BBa_I20260 using PCR and restriction digests showed that the insert was not present in the vector, and the conversion to BioBrick format ultimately unsuccessful. The procedure as described above was repeated multiple times without success. Tri-parental conjugation of unmodified pRL 1383a was inconclusive in all target organisms.</p><br />
<br />
<p class="class">In an effort to troubleshoot this vector, several different approaches were taken. First, the ligation was repeated with varying concentrations of insert (10X, 2X) in an attempt to account for the impact of the large vector size on the ligation reaction. These ligations yielded similar results to reactions done at calculated concentrations. A Blunt-end ligation using a Klenow fragment was also performed. This was repeated, both attempts without success. The BBa_I20260 PCR product with BamHI/HindIII ends was ligated into another vector in an attempt to test the insert’s ability to be cut with the restriction enzymes. This ligation did not indicate the presence of the insert, suggesting that the problem lies with the vector or primers. The primers were tested and found viable on another insert, with similar testing of restriction enzymes to show functionality. The primers and enzymes were operating as intended, but new enzymes were ordered for more experimental certainty. The insert was then digested only with HindIII, and left in a ligation reaction. The outcome of this ligation was not of the desired length. This was repeated, and the same result obtained. While there is some suggestion that the BioBrick insert may not be functioning, the ambiguous results of tri-parental mating with unmodified pRL1383a suggests that the vector may be damaged or misunderstood.</p><br />
<br />
<p class="class">Testing the ligation of PCPP33 and BBa_I20260 also proved unsuccessful. Restriction digests using BioBrick standard pieces failed to yield an insert. Tri-parental mating of this vector proved successful in all organisms that we tested. All organisms yielded colonies on tetracycline plates, suggesting presence of the plasmid. Further testing by plasmid extraction and gel analysis will be done to conclusively determine presence of the plasmid. <br />
</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/7/77/R_spaeroides_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/1/1e/P_putida_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/d/da/Synechocystis_PCPP33.JPG" align = "left" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 4</b> Results of the tri-parental mating between pCPP33 and R. <i>sphaeroides</I>, P. <i>putida</i>, and Synechocystis sp., respectively. Each plate is shown alongside a negative control <br />
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Experiments: Secretion<br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20><br />
Methods for Constructing BioBrick Parts<br />
</font><br></b><br />
<br />
<p class="class"><br />
One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length. <br />
<br />
<p class="class"><br />
Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.</p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Signal Peptides:</font></b><br><br />
<p class="class"><br />
To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.</p><br />
<p class="class"><br />
The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Phasin:</font></b><br><br />
<p class="class"><br />
The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis (LINK TO PROTOCOLS PAGE) (CTGCAG CTTCAG). Sequencing confirmed that this site was successfully removed. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>GFP:</font></b><br><br />
<p class="class"><br />
Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported. Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator. </p><br />
<br />
<p class="class"><br />
A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure X shows GFP- Top10 <i>E. coli</i> colonies (left) and unfused GFP+ Top10 <i>E. coli</i> colonies (right). This figure shows that the GFP construct is functional and easily detectable.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/93/GFPglowingUSU.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Plate with GFP- cells (right) next to plate with GFP+ cells(left)<br />
</div><br />
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<br><b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Bioplastic Production:</font></b><br><br />
<p class="class">A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. <i>E. coli</i> harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure X. The observed peaks at 1.24 ppm, 2.54 ppm, and 5.2 ppm correspond with those observed in standard polyhydroxyalkanaote samples.</p><br />
<Br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/4/43/NMRusu.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Proton NMR spectra for PHB production in recombinant <i>E. coli</i><br />
</div><br />
<br><br />
<p class="class">To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>SDS-PAGE Analysis</font></b><br><br />
<p class="class">Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged (see Figure X). Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure X. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/d/d1/GFP_gel.png"" align = "middle" height="400" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing a strong band corresponding to GFP<br />
</div><br />
<br><br />
<p class="class">The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection, as shown in Figure X. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/3/3e/PHB_gel.png"" align = "middle" height="250" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing the presence of phasin protein in supernatant samples (third well from left)<br> next to supernatant from an <i>E. coli</i> sample without a phasin-producing construct.<br />
</div><br />
<Br><br />
<br />
<p class="class">Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Figure X shows the apparatus used to transfer proteins onto PVDF paper. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies. </p><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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Experimental Section: Approach for BioBrick Compatibility<br />
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Converting Broad-Host Vectors into a BioBrick-Compatible Format<br />
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<p class="class">Two Broad-host range vectors were used in this study; pRL1383a and PCPP33. To convert these vectors into BioBrick-compatible format, the four standard BioBrick sites EcoRI, XbaI, SpeI, and PstI needed to be inserted into the multiple cloning site. For pRL1383a, common BioBrick primers VR and VF2 were also included to allow the use of PCR in amplifying the BioBrick parts.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/8/82/PRL1383A_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 2</b> Plasmid map of pRL1383a <br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/a/af/PCPP33_Plasmid_Map.jpg"" align = "middle" height="300" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 3</b> Plasmid map of pCPP33 <br />
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<p class="class">Apart from being shown effective in the Synechosystis PCC 6803 (Marraccini 1993), pRL1383a is an ideal candidate for use as a BioBrick-compatible broad-host range vector because the BioBrick restriction sites are absent within the vector sequence. To convert pRL1383a into a BioBrick format, the existing multiple cloning site, which is flanked by a BamHI site and a HindIII site, was utilized. First, modified primers were synthesized from BioBrick primers VR and VF2. These primers were modified by adding extra nucleotides to insert the desired restriction enzyme sites into the PCR product. A BamHI site was added to 5’ end of the forward primer (VF2) and a HindIII site was added to the 5’ end of the reverse primer (VR). These primers were used to amplify an existing, tested BioBrick part by PCR. For this purpose, we selected BBa_I20260 because it does not contain BamHI or HindIII sites, and successful ligation is readily testable as it is a GFP -producing construct. The addition of IPTG is typically necessary to induce GFP production in this particular device. However, when using Top10 <i>E. coli</i> cells it is produced continuously because these cells lack a lac repressor (insert invitrogen link). After cutting the vector at the multiple cloning site using BamHI and HindIII, the BioBrick insert obtained by PCR with modified ends was ligated into the backbone. The vector was then transformed using Top10 One Shot® chemically competent <i>E. coli</i> and tested for successful insertion using PCR and restriction digests.</p><br />
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<p class="class">Another broad host range vector, pCPP33, previously shown effective in Pseudomonas Putida,was standardized using similar methods. While the complete sequence of this plasmid is not available, it was shown that there are no BioBrick restriction sites outside the multiple cloning site (Figure 3). The multiple cloning site of this vector is flanked by EcoRI and HindIII. This allowed the PCR product of BBa_I20260 to again be used by cutting with HindIII and EcoRI restriction enzymes. Restriction digests and gel analysis were used to test for the insert.</p><br />
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Broad Host Conjuation<br />
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<p class="class">In order to transfer a vector of interest using conjugation, the <i>tra</i> gene (contained in what we will refer to as a transfer plasmid, or helper plasmid) must be expressed in order to initiate the conjugation process. This plasmid codes for genes which, when expressed, form pili on the cell surface, which in turn initiate conjugation (Heinemann 1989). This plasmid may be present in one of three different procedures:</p><br />
<ul><br />
</li><li><b>Hfr strain</b> – The <i>tra</i> operon is many times contained in an episome, which can incorporate itself into the cell genome. These resultant Hfr strains will often begin the transfer of their own DNA, both plasmid and genomic. Due to the transfer of the genomic DNA, these strains are referred to as high frequency recombinant (Hfr) strains.<br />
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</li><li><b>Biparental (normal) Conjugation</b> – Cells containing the <i>tra</i> genes, often labeled as F-positive (F+) due to the F-plasmid, a well-known transfer plasmid, can express the transfer genes necessary for conjugation to occur. When a vector of interest and a transfer plasmid are of different incompatibility groups, they may both be transformed into the same cell, and conjugation may take place between the F+ donor cell and the recipient cell<br />
<br />
</li><li><b>Triparental Mating</b> – In the case where the transfer plasmid and the vector of interest are of the same incompatibility group, the two plasmids may not stably coexist (Heinemann 1989). In this case, two separate cells containing the transfer gene (the helper cell) and the vector (the donor cell) must be used in conjugation. The helper cell will assist the donor cell in the transfer of its mobilizable plasmid to the recipient cell. This method circumvents some of the barriers that may prevent the transfer of plasmids.<br />
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<p class="class">For our project, we chose to use the triparental mating procedure for the transmission of our vector. While not being the most efficient method, it circumvents possible barriers and intermediate steps.</p><br />
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<p class="class">Because of the use of three different cells in our transformation procedure, the selection criteria for each component needed to be unique. In addition, we selected helper plasmids which had been known to work with the intended recipient cell.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/4/4a/PCPP33_tri-p_table.png" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Table 1</b> Components and selection criteria used in conjugation with the broad-host vector PCPP33 <br />
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<div align="center"><img src="https://static.igem.org/mediawiki/igem.org/f/f2/PRL1383A_tri-p_table.png" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Table 2</b> Components and selection criteria used in conjugation with the broad-host vector PRL1383A <br />
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Results<br />
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<p class="class">Testing the ligation of pRL1383a and BBa_I20260 using PCR and restriction digests showed that the insert was not present in the vector, and the conversion to BioBrick format ultimately unsuccessful. The procedure as described above was repeated multiple times without success. Tri-parental conjugation of unmodified pRL 1383a was inconclusive in all target organisms.</p><br />
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<p class="class">In an effort to troubleshoot this vector, several different approaches were taken. First, the ligation was repeated with varying concentrations of insert (10X, 2X) in an attempt to account for the impact of the large vector size on the ligation reaction. These ligations yielded similar results to reactions done at calculated concentrations. A Blunt-end ligation using a Klenow fragment was also performed. This was repeated, both attempts without success. The BBa_I20260 PCR product with BamHI/HindIII ends was ligated into another vector in an attempt to test the insert’s ability to be cut with the restriction enzymes. This ligation did not indicate the presence of the insert, suggesting that the problem lies with the vector or primers. The primers were tested and found viable on another insert, with similar testing of restriction enzymes to show functionality. The primers and enzymes were operating as intended, but new enzymes were ordered for more experimental certainty. The insert was then digested only with HindIII, and left in a ligation reaction. The outcome of this ligation was not of the desired length. This was repeated, and the same result obtained. While there is some suggestion that the BioBrick insert may not be functioning, the ambiguous results of tri-parental mating with unmodified pRL1383a suggests that the vector may be damaged or misunderstood.</p><br />
<br />
<p class="class">Testing the ligation of PCPP33 and BBa_I20260 also proved unsuccessful. Restriction digests using BioBrick standard pieces failed to yield an insert. Tri-parental mating of this vector proved successful in all organisms that we tested. All organisms yielded colonies on tetracycline plates, suggesting presence of the plasmid. Further testing by plasmid extraction and gel analysis will be done to conclusively determine presence of the plasmid. <br />
</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/7/77/R_spaeroides_PCPP33.JPG" align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/1/1e/P_putida_PCPP33.JPG" align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /><img src="https://static.igem.org/mediawiki/2009/d/da/Synechocystis_PCPP33.JPG" align = "left" height="160" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 4</b> Results of the tri-parental mating between pCPP33 and R. <i>sphaeroides</I>, P. <i>putida</i>, and Synechocystis sp., respectively. Each plate is shown alongside a negative control <br />
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Experiments: Secretion<br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20><br />
Methods for Constructing BioBrick Parts<br />
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<p class="class"><br />
One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length. <br />
<br />
<p class="class"><br />
Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.</p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Signal Peptides:</font></b><br><br />
<p class="class"><br />
To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.</p><br />
<p class="class"><br />
The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Phasin:</font></b><br><br />
<p class="class"><br />
The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis (LINK TO PROTOCOLS PAGE) (CTGCAG CTTCAG). Sequencing confirmed that this site was successfully removed. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>GFP:</font></b><br><br />
<p class="class"><br />
Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported. Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator. </p><br />
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<p class="class"><br />
A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure X shows GFP- Top10 <i>E. coli</i> colonies (left) and unfused GFP+ Top10 <i>E. coli</i> colonies (right). This figure shows that the GFP construct is functional and easily detectable.</p><br><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/93/GFPglowingUSU.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
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<b>Figure 2.</b> Plate with GFP- cells (right) next to plate with GFP+ cells(left)<br />
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<br><b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Bioplastic Production:</font></b><br><br />
<p class="class">A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. <i>E. coli</i> harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure X. The observed peaks at 1.24 ppm, 2.54 ppm, and 5.2 ppm correspond with those observed in standard polyhydroxyalkanaote samples.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/4/43/NMRusu.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Proton NMR spectra for PHB production in recombinant <i>E. coli</i><br />
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<p class="class">To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>SDS-PAGE Analysis</font></b><br><br />
<p class="class">Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged (see Figure X). Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure X. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/d/d1/GFP_gel.png"" align = "middle" height="400" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing a strong band corresponding to GFP<br />
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<br><br />
<p class="class">The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection, as shown in Figure X. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/3/3e/PHB_gel.png"" align = "middle" height="250" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing the presence of phasin protein in supernatant samples (third well from left)<br> next to supernatant from an <i>E. coli</i> sample without a phasin-producing construct.<br />
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<p class="class">Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Figure X shows the apparatus used to transfer proteins onto PVDF paper. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies. </p><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
</tr><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
</tr><br />
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<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>BioBricks without Borders:<br />
</b></i></font><br />
<br><br />
<font size ="2.5" face="tahoma, sans-serif, verdana" color=#009900><br />
Investigating a multi-host BioBrick vector and secretion of cellular products</font><br />
<HR><br />
<br />
<p class = "class"> <br />
The aim of the Utah State University iGEM project is to develop improved upstream and downstream processing strategies for manufacturing cellular products using the standardized BioBrick system. First, we altered the broad-host range vector pRL1383a to comply with BioBrick standards and enable use of BioBrick constructs in organisms like Pseudomonas putida, Rhodobacter sphaeroides, and Synechocystis PCC6803. This vector will facilitate exploitation of advantageous characteristics of these organisms, such as photosynthetic carbon assimilation. Following expression, product recovery poses a difficult and expensive challenge. Downstream processing of cellular compounds, like polyhydroxyalkanoates (PHAs), commonly represents more than half of the total production expense. To counter this problem, secretion-promoting BioBrick devices were constructed through genetic fusion of signal peptides with protein-coding regions. To demonstrate this, the secretion of PHA granule-associated proteins and their affinity to PHA was investigated. Project success will facilitate expression and recovery of BioBrick-coded products in multiple organisms.<br />
</P> </td></tr><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/ProjectTeam:Utah State/Project2009-10-22T02:00:57Z<p>Liblint: </p>
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</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br /><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>BioBricks without Borders:<br />
</b></i></font><br />
<br><br />
<font size ="2.5" face="tahoma, sans-serif, verdana" color=#009900><br />
Investigating a multi-host BioBrick vector and secretion of cellular products</font><br />
<HR><br />
<br />
<p class = "class"> <br />
The aim of the Utah State University iGEM project is to develop improved upstream and downstream processing strategies for manufacturing cellular products using the standardized BioBrick system. First, we altered the broad-host range vector pRL1383a to comply with BioBrick standards and enable use of BioBrick constructs in organisms like Pseudomonas putida, Rhodobacter sphaeroides, and Synechocystis PCC6803. This vector will facilitate exploitation of advantageous characteristics of these organisms, such as photosynthetic carbon assimilation. Following expression, product recovery poses a difficult and expensive challenge. Downstream processing of cellular compounds, like polyhydroxyalkanoates (PHAs), commonly represents more than half of the total production expense. To counter this problem, secretion-promoting BioBrick devices were constructed through genetic fusion of signal peptides with protein-coding regions. To demonstrate this, the secretion of PHA granule-associated proteins and their affinity to PHA was investigated. Project success will facilitate expression and recovery of BioBrick-coded products in multiple organisms.<br />
</P> </td></tr><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/SecretionTeam:Utah State/Secretion2009-10-22T01:55:02Z<p>Liblint: </p>
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</tr><br />
<tr><br />
<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br />
</tr><br />
<br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
</tr><br />
<tr><br />
<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
</tr><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. coli MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. coli transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. coli or photoautotrophic PHA-producers R. sphaeroides and Synechocystis PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure X depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant E. coli do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure X depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. coli K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure X. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative E. coli (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure X illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure X shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of E. coli (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in E. coli harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in E. coli (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 1 depicts the GFP barrel structure.</p><br />
<br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="200" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> The GFP Barrel Structure<br />
</div><br />
<br />
<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. coli, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. coli MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. coli transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. coli or photoautotrophic PHA-producers R. sphaeroides and Synechocystis PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure X depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant E. coli do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure X depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. coli K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure X. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative E. coli (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure X illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure X shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of E. coli (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in E. coli harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in E. coli (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 1 depicts the GFP barrel structure.</p><br />
<br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="200" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> The GFP Barrel Structure<br />
</div><br />
<br />
<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. coli, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/SecretionTeam:Utah State/Secretion2009-10-22T01:53:33Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
<a href="#references">References</a><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Experiments"><font size = 4>EXPERIMENTS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. coli MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. coli transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. coli or photoautotrophic PHA-producers R. sphaeroides and Synechocystis PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure X depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant E. coli do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure X depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. coli K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure X. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative E. coli (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure X illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure X shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of E. coli (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in E. coli harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in E. coli (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 1 depicts the GFP barrel structure.</p><br />
<br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="200" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> The GFP Barrel Structure<br />
</div><br />
<br />
<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. coli, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/SecretionTeam:Utah State/Secretion2009-10-22T01:50:24Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. coli MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. coli transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. coli or photoautotrophic PHA-producers R. sphaeroides and Synechocystis PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure X depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant E. coli do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure X depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. coli K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure X. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative E. coli (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure X illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure X shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of E. coli (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in E. coli harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor, as shown in Part C of Figure X. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in E. coli (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 1 depicts the GFP barrel structure.</p><br />
<br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="200" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> The GFP Barrel Structure<br />
</div><br />
<br />
<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. coli, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/SecretionTeam:Utah State/Secretion2009-10-22T01:49:37Z<p>Liblint: </p>
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Secretion: Bioplastics, Phasin, and GFP<br />
</font></b></i> <hr><br />
<p class="class"><br />
Recovery of cellular products is often a difficult and expensive challenge. As much as 80% of protein production costs are attributable to downstream processing (Hearn and Acosta, 2001). Likewise, the separation and purification cost for non-protein products, like polyhydroxyalkanaotes (PHAs) are significant and commonly represent more than half of the total process expense (Ling, 1998; Jung, 2005). </p><br />
<p class="class"><br />
Polyhydroxyalkanoates comprise a class of polyesters that are generated by a variety of microorganisms (Anderson and Dawes, 1990; Doi, 1990). These bioplastic compounds are intracellularly accumulated and stored as a reserve of carbon, energy, and reducing power in response to an environmental stress or nutrient limitation (Lee, 1996). Polyhydroxybutyrate (PHB) is the most common form of PHA. PHAs have comparable material properties to conventional plastics, like polypropylene, but are fully biodegradable and renewable (Steinbüchel and Füchtenbusch, 1998). As a result, PHAs are of particular interest as a sustainable source of non-petrochemically derived thermoplastics for use in an assortment of commercial and medical applications (Madison and Huisman, 1999).</p><br />
<br />
<p class="class">Costs associated with the PHA manufacturing process have limited the widespread application of the bioplastic material (Lee, 1996). Economic analyses for industrial scale PHA production place the cost of PHAs at about $4-5/kg (Choi, 1997; Choi, 1999). In contrast, the average cost of petrochemically-derived plastic lies between $0.62-0.96/kg (Steinbüchel and Füchtenbusch, 1998). This significant discrepancy in expense is largely attributable to downstream processing. Traditional methods involving the use of solvents, enzymatic digestion, or mechanical disruption are expensive and impractical for industrial-scale recovery (Jung, 2005). As a result, the development of alternative methods for PHA recovery is necessary.</p><br />
<br />
<p class="class">Genetic engineering strategies have been used in attempts to simplify PHA recovery and eliminate the need for mechanical or chemical cellular disruption. Jung et al. (2005) used recombinant E. coli MG1655 harboring PHA biosynthesis genes from C. necator to instigate spontaneous autolysis of the cell wall. Up to 80% of the cells in culture released PHA granules, which were subsequently recovered using centrifugation and washing with distilled H2O (Jung, 2005). Resch et al. (1998) used recombinant PHA-producing E. coli transformed with the E-lysis gene of bacteriophage PhiX174 from plasmid pSH2. Amorphous PHB in is pushed out of the cell through an E-lysis tunnel structure, which is an opening in the cell envelope (Resch, 1998). In this procedure, the osmotic pressure difference between the cytoplasm and the culture medium provides the driving force for PHA movement into the extracellular medium. The PHA is then recovered by centrifugation or through the addition of divalent cations (Resch, 1998). Although these methods use genetic means to bring about cellular disruption, these mechanisms still require cellular death and fail to promote a continuous production system. </p><br />
<br />
<p class="class">Recently, extracellular deposition of PHA granules was observed in a mutant strain of Alcanivorax borkumensis SK2, which is a marine bacterium that uses hydrocarbons as its source of carbon and energy (Sabirova, 2006). This finding by Sabirova et al (2006) is the first account of PHA accumulation outside of the cell (Prieto, 2007). However, the mechanism by which this deposition occurs is unknown (Sabirova, 2006; Prieto, 2007). A defined system for microbial excretion of PHAs has yet to be created. Such a system would be of value due to the potential to optimize and introduce the mechanism into other organisms with advantageous characteristics, such as fast-growing E. coli or photoautotrophic PHA-producers R. sphaeroides and Synechocystis PCC6803. </p><br />
<p class="class">PHA-associated proteins, called phasins, strongly interact with the PHA granule surface (York, 2001; Maehara, 1999). Accordingly, PHA recovery may be possible by tagging the phasin protein for translocation. Specifically, the Silver fusion Biobrick standard can be used to create constructs in which a targeting signal peptide sequence is genetically fused to the phasin protein (Phillips, 2006). Fusing a signal peptide to a protein promotes export of the complex out of the cytoplasm (Choi, 2004; Mergulhão, 2005). The interaction of phasin with PHA is required for secretion-based granule recovery because PHA is a non-proteinaceous compound produced by the action of three enzymes (Suriyanmongkol 2007; Verlinden 2007). Consequently, the signal peptide cannot be directly attached PHA granules. The phasin protein with attached signal peptide binds to PHA granules, thereby creating a PHA-phasin-signal peptide complex that may be recognized by the cell for export. Figure X depicts this export process in general terms. Green fluorescent protein (GFP) translocation has been documented (Barrett, 2003; Santini, 2001; Thomas, 2001). Due to its ease of detection, studying GFP in parallel with phasin secretion mechanisms could provide a framework for determining the functionality of secretion systems.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/2/25/Bioplasticscheme.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Schematic for bioplastic recovery by secretion<br />
</div><br />
<br><br />
<p class="class"><br />
Secretion-based product recovery mechanisms hold great potential to improve the economics of industrial-scale production systems. In addition to reduced downstream processing requirements, secretory production has additional benefits, such as potentially improved product stability and solubility (Mergulhão, 2005). Recombinant E. coli do not typically secrete high levels of proteins and functionality of proteins secretion is difficult to predict (Sandkvist, 1996; Choi, 2004). Accordingly, a trial-and-error approach with different combinations of signal peptides and promoters is recommended for any given protein, and will be discussed in more detail in subsequent sections (Choi, 2004). <br />
</p></p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Principles of Recombinant Protein Secretion<br />
</font></b></i><br />
<br />
<p class="class"><br />
The functionality of protein secretion mechanisms is affected by the structural differences between gram-positive and gram-negative organisms (Desveaux, 2004; Sandkvist, 1996). Gram-positive species have a solitary cytoplasmic membrane, which effectively means that protein membrane translocation is equivalent to secretion in these species (Pugsley, 1993). Alternatively, gram-negative organisms have both an inner and outer membrane that proteins must cross for secretion. Accordingly, proteins can either be exported into the periplasmic space or secreted fully into the extracellular medium (Pugsley, 1993). </p><br />
<p class="class"><br />
There are five pathways observed for secretion of recombinant proteins in gram-negative prokaryotes, numbered I through V (Desvaux, 2004; Mergulhão, 2005). While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure (Koster, 2000). Types I and II are the most common pathways for recombinant protein secretion (Mergulhao, 2005) and will be discussed here. </p><br />
<p class="class">Type I secretion is a single-step translocation of protein across both inner and outer membranes. (Binet, 1997). The constituents of this system include inner membrane proteins HlyB and HlyD, as well as the TolC outer membrane protein (Mergulhão, 2005; Desveax, 2004). These three proteins interact to form a channel that spans the periplasm (Mergulhão, 2005). Appending the last 42-60 amino acids of the HlyA protein C-terminus to the C-terminus of a recombinant protein targets the protein for secretion (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The HlyA signal sequence binds to the channel complex, resulting in ATP hydrolysis by HlyB to drive protein secretion (Gentschev, 2003). Proteins as large as 4000 amino acids can be secreted through the type I channel, which has an internal diameter of 3.5 nm and a length of 14 nm (Sapriel, 2003; Fernandez and de Lorenzo, 2001). Unlike in the Type II pathway, the signal peptides of Type I secretion remain attached to the protein after export out of the cytoplasm (Blight and Holland, 1994). Figure X depicts the secretion of a protein with a C-terminal fused HlyA signal peptide by Type I secretion (Mergulão, 2005). <br />
<br><br />
</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/e/ed/FigureHlyATypeI.png"" align = "middle" height="150" style="padding:.5px; border-style:solid; border-color:#999" alt="HlyA" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> HlyA Type I Secretion Pathway<br />
</div><br />
<br><br />
<p class="class"><br />
The type II secretion pathway is a two-step process. The cytoplasmic protein must first be exported into the periplasm through the action of a translocase. Specifically, the Sec and Twin-arginine translocation (TAT) machinery facilitate protein movement across the inner membrane and will be discussed in detail in the next section. After entering the periplasm, the protein can be translocated into the extracellular medium through the action of a secreton, which is a 12-16 core protein complex present in many gram-negative strains, such as E. coli K-12 (Cianciotto, 2005). Although the secreton functionality is not completely understood, it is known that protein conformational changes are necessary for this process to be carried out (Mergulhão, 2005; Sandkvist, 2001).</p> <br />
<br />
<p class="class"><br />
Translocation of cellular products into the periplasm is advantageous over cytoplasmic production because recovery of periplasmic products is relatively simpler (Mergulhão, 2005). There are additional mechanisms for recovering periplasmic proteins if the secreton machinery is either not present in the host strain or incompatible with the protein of interest. These mechanisms are depicted in Figure X. L-form and Q-cells are mutant strains that have a weakened outer membrane, which allows for some proteins to leak into the extracellular medium (Mergulhão, 2005). However, these organisms have reduced growth rates and are not ideal candidates for general cellular production. The permeability of the outer membrane may be enhanced mechanically, such as by application of ultrasound, or through chemical treatment, such as through addition of Triton X-100 or 2% glycine (Kaderbhai, 1997; Choi, 2004). As another example, enzymatic digestion with lysozyme breaks the outer membrane to release periplasmic proteins (Shokri, 2003). Yet another alternative involves coexpression of genes, such as kil, out, and tolAIII, that cause cellular lysis and subsequent release of recombinant proteins (Choi, 2004; Mergulhão, 2005). The downside to these alternatives is the weakening of cell integrity.<br />
</p><br><br />
<br />
<b><i><font size="3" face="Helvetica, Arial, San Serif" color =#000033><br />
Cytoplasmic Membrane Translocation in the Type II Pathway<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Several membrane-associated components mediate translocation of proteins across the inner membrane of gram-negative E. coli (Luirink, 2004). This machinery includes translocases, ATPases, and accessory proteins (Luirink, 2004; Veenendaal, 2004). The Sec pathway and the TAT system are the two general mechanisms by which proteins are transported into the periplasm, with the Sec-translocon providing most common export route (Luirink, 2004; Veenendaal, 2004). Within the Sec-dependent category, proteins are exported either via the SecB-dependent pathway or by the action of the signal recognition particle (SRP). The attachment of a short sequence, called a signal peptide, to the N-terminus of a protein is generally necessary for targeting proteins to any of the three translocation pathways (Luirink, 2004; Choi, 2004; Mergulhão, 2005). </p><br />
<br />
<p class="class">In the Sec pathway, SecA is attached peripherally to the inner membrane and drives peptide translocation through ATPase activity (van der Does, 2004). Integral membrane proteins SecY and SecE form the core of the Sec translocon, and SecG interacts with this core to form a multimeric protein complex, SecYEG (Veenendaal, 2004). This complex functions as a protein-conducting channel for both post-translational and co-translational protein export (Luirink, 2004; Veenendaal, 2004). Interestingly, the SecYEG translocon can be found in all domains of life, reiterating the prevalence and importance of this mechanism for protein export (Cao, 2002). </p><br />
<br />
<p class="class">A SecB-dependent mechanism is used by gram-negative species to target post-translational periplasmic and outer membrane proteins to the Sec-translocon (Luirink, 2004). Of the three translocation routes, the Sec-B pathway is the most common for recombinant protein export (Mergulhão, 2005). First, a trigger factor binds to the preprotein as it leaves a ribosome (Luirink, 2004; Mergulhão, 2005). Next, the unfolded protein is recognized and bound by the SecB chaperone protein and directed to SecA, where ATP hydrolysis provides the force to drive the protein through the SecYEG translocase into the periplasm (Mergulhão, 2005). In co-translational protein export, a signal recognition particle (SRP) identifies and interacts with the signal sequence of the nascent protein as it is exiting the ribosome to the Sec-translocon (Luirink, 2004; von Heijne, 1996; Mergulhão, 2005). </p><br />
<br />
<p class="class"><br />
The TAT system is used to export folded proteins into the periplasmic space (Choi, 2004). Like the Sec-dependent pathways, specific N-terminal signal peptide sequences target a protein for export by the TAT machinery. Although similar, TAT signal peptides differ from those that target proteins to the Sec machinery. TAT signal peptides contain a conserved sequence of seven amino acids, (S/T)-R-R-x-F-L-K, at the interface between the N- and H-regions, where x represents a polar amino acid (Berks, 2000; Palmer, 2004). The twin-arginine residues are consistently present in TAT signal peptides, and the occurrence of the other amino acids is greater than 50% (Berks 1996, Berks 2000, Palmer, 2004). Figure X illustrates the mechanism for protein export by the Sec and TAT pathways.</p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/91/FigureSecTAT.png"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Mechanism of protein translocation by Sec and Tat<br />
</div><br />
<br><br />
<br />
<p class="class"><br />
Whether a protein is targeted to the SecB, SRP, or TAT pathways is largely dependent on the characteristics of the attached signal peptide (Mergulhão, 2005; van der Does, 2004; Luirink, 2004). For example, the hydrophobicity of the signal peptide plays a role in designating which route will be used for protein export (Berks, 2000; Luirink, 2004). The affinity of a signal sequence to the SRP increases as the number of hydrophobic residues in the H-domain of the signal peptide (Valent, 1997). The trigger factor of the SecB pathway recognizes slightly less hydrophobic sequences in the signal peptide and consequently prevents binding by the SRP. Lastly, TAT pathway signal sequences are the most hydrophilic in the H-domain (Berks, 2000). Moreover, increasing H-domain hydrophobicity of TAT signal sequences can even divert a protein typically translocated via the TAT pathway to the Sec translocon (Berks, 2000; Cristobal, 1999). The mature region of the protein may also play a role in pathway targeting, particularly in regard to the SecB mechanism (Luirink, 2004). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Signal Peptides<br />
</font></b></i> <br />
<p class="class"><br />
Signal peptides consist of about 15-30 amino acids and are generally required to direct a secretory protein to the translocons of the cytoplasmic membrane (Pugsley, 1993; Choi, 2004; Luirink, 2004). Despite overall sequence variability, structural similarities exist between different signal peptides, including a positively-charged 2-10 amino acid N-region, a hydrophobic core H-region, and a neutral C-domain of about 6 residues (Pugsley, 1993; Molhoj, 2004; Berks, 2000). The C-domain conforms to the -3, -1 rule in which amino acids with short and neutral side-chains, such as alanine, are required in positions -3 and -1 of the sequence (Choi, 2004; von Heijne, 1984). A signal peptidase interacts with a cleavage recognition site within the C-domain to release the protein into the periplasmic space (Luiritz, 2004; Choi, 2004). The absence or mutation of the cleavage site can lead to the targeted protein remaining fixed to the inner membrane (Luiritz, 2004). Figure X shows the typical composition of a signal peptide sequence.</p><br><br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/f/f2/Signal_peptide.png"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> Typical signal peptide sequence<br />
</div><br />
<br />
<br><br />
<p class="class"><br />
A small signal sequence is typically necessary for all translocation pathways. However, certain protein-coding sequences can be secreted without having an attached signal sequence due to the presence of additional targeting information within the sequence (Luiritz, 2004). Additionally, an attached signal sequence does not guarantee export of a protein, which further suggests that information in the protein sequence itself can affect secretion efficiency (Luiritz, 2004). However, the fusion of a signal sequence to a recombinant protein can lead to export of a previously non-secretable protein. There are many reported examples of recombinant protein translocation through signal sequence gene fusion. For example, fusion with the Tat-dependent signal peptide TorA allowed for export of folded GFP into the periplasm of E. coli (Palmer, 2004; Barrett, 2003; Santini, 2001; Thomas, 2001). </p><br />
<br />
<p class="class"><br />
Two factors that affect protein export are the positive charge of the N-terminus of the signal peptide and the charge of the N-terminus of the recombinant protein (Akita 1990). Akita et. Al (1990) determined that increasing the positive charge of the signal peptide N-terminus not only enhances the interaction with SecA protein, but also reduces the requirements of SecA ATPase activity for translocation. Therefore, a higher net positive N-terminus charge improves the rate of protein translocation (Mergulhão, 2005). For the recombinant protein, the charge of the N-terminus also affects protein secretion. A net positive charge within the first five amino acids near the C-domain cleavage site of the signal sequence can reduce protein export by as much as 50-fold because the charge inhibits the protein from entering the lipid bilayer (Schatz, 1990). </p><br />
<br />
<p class="class"><br />
Although factors like hydrophobicity and charge are known to affect protein export, there are few available guidelines for selecting a proper signal peptide for any given protein (Choi, 2004). It is advised to carry out investigation of recombinant protein secretion by trial-and-error with different host strains and signal peptides (Choi, 2004). The mechanisms of protein secretion are complicated and many obstacles can inhibit the process. Some commonly observed problems include incomplete translocation, degradation of recombinant protein by proteases, formation of inclusion bodies, and inefficiency of secretion machinery (Mergulhão, 2005; Choi, 2004). Optimization of the secretion efficiency requires balancing the promoter strength and gene copy number so as not to overwhelm the system (Mergulhão, 2005). Lastly, some proteins may simply be unsuitable for secretion due to their size or sequence (Koster, 2000). </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Phasin<br />
</font></b></i> <br />
<br />
<p class="class"><br />
Phasin (PhaP) is a low-molecular weight protein that plays a role in PHA granule formation by physically binding to the PHA granule surface (York, 2001). The specific purpose of phasin production is not completely understood (York 2002), although some of the affects of the phasin/PHA interaction have been studied. York et al (2001) determined that the production of phasin is dependent on PHA accumulation. Specifically, it is suggested that phasin expression requires the presence of PHA synthase (York, 2001). Maehara et al (1999) observed that the level of PHA accumulation substantially decreases and the size of PHA granules increases when phasin is either absent or regulated by a repressor, PhaR. Therefore, PHA production levels are enhanced in the presence of phasin due to an increased granule surface-to-volume ratio (York 2001; Maehara 1999). </p><br />
<br />
<p class="class"><br />
In addition to reducing PHA granule size, other functions of phasin have been proposed. In the absence of phasin, other proteins can bind to the granule surface (Maehara, 1999). Therefore, phasins may function to inhibit attachment of other proteins to the PHA surface that could cause defects in granule formation (York 2001; Maehara, 1999). Lastly, it is suggested that phasins promote PHA synthesis through an interaction with PHA synthase (York, 2001). </p> <br />
<br />
<p class="class"><br />
Due to their physical interaction with the PHA granule, phasins can be used in recombinant protein purification (Banki, 2005), or PHA recovery as this project is investigating. For protein purification, genetic fusion of a protein product, a self-splicing element called an intein, and phasin can be used (Banki, 2005). The genetically-fused protein is produced in E. coli harboring the PHB production genes (Banki, 2005). The phasin protein binds to the surface of the PHB granule, and a cleavage-inducing buffer stimulates the release of the product protein into the soluble fraction of the solution (Banki, 2005). </p><br />
<br />
<p class="class"><br />
For this procedure, PHB is released and proteins are recovered only after the cell lysed, which is not ideal. However, the system provides evidence that the phasin/PHA interaction may be exploited for improving production processes and that genetic fusion of other elements with phasin does not inhibit binding to PHA (Banki, 2005). The fusion of phasin with a signal peptide, which is a sequence that tags a protein for secretion, could result in a signal peptide/phasin/PHA complex that is recognized by cell for transmembrane export. </p><br />
<br />
<p class="class"><br />
The recovery of PHA granules via secretion of a signal peptide/phasin/PHA complex may be inhibited due to the size of PHA granules. However, the binding of phasins decreases PHA molecular weight and encourages the formation of numerous, small granules (Maehara, 1999). Though the actual size of PHA granules varies, Maehara et al (1999) observed spherical granules approximately 20 – 60 nm in diameter in the presence of phasin and absence of the PhaR repressor, as shown in Part C of Figure X. This indicates that enhanced production of phasin may further reduce granule size, which may make PHAs more suitable for export. </p><br />
<br />
<b><i><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
Green Flourescent Protein<br />
</font></b></i> <br />
<br />
<p class="class"><br />
GFP is a commonly used reporter of gene regulation. It is expressed in many bioluminescent jellyfish naturally (Shimomura, 1962). Its value in the academic and biotechnology industry was recognized after successful cloning and expression in E. coli (Chalfie, 1994). Purified GFP, composed of 238 amino acids, absorbs blue light (395 nm) and emits green light (Chalfie, 1994). The detection of intracellular GFP is not limited by the availability of substrates, but requires only irradiation by near UV or blue light (Chalfie, 1994). However, to ease the process of GFP detection for many organisms, a stronger whole cell fluorescence signal is desirable. Figure 1 depicts the GFP barrel structure.</p><br />
<br />
<br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/6/60/GFpbarrel.jpg"" align = "middle" height="50" style="padding:.5px; alt="signal peptide" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure X.</b> The GFP Barrel Structure<br />
</div><br />
<br />
<p class="class">Many mutant forms of GFP have been created which improve fluorescence photostability and ultimately the ability of GFP to function as a practical reporter. The cycle 3 mutant developed by Crameri et al. (1996) is of special interest because it produces a fluorescence signal 45-fold greater than wild-type GFP. The developed GFP possesses three point mutations of the wild-type GFP. These mutations do not affect the chromophore itself, but reside in the surrounding barrel of the GFP protein. In E. coli, due to its hydrophobic nature, most of the wild-type GFP gathers to form inclusion bodies that limit the ability of blue light to provide the necessary excitation energy to activate fluorescence (Crameri , 1996). The three point mutations in the cycle 3 mutant, have no effect on excitation and emissions maxima, but create a more hydrophilic GFP less prone to form inclusion bodies. The soluble mutant is easily activated by a UV light box or light wand common in the laboratory creating an immediate, practical reporter protein. Furthermore, fusions onto amino- or carboxy-termini of GFP do not inhibit fluorescence, which makes GFP an ideal candidate for fusion studies (LaVallie, 1995).</p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/NotebookTeam:Utah State/Notebook2009-10-22T01:47:30Z<p>Liblint: </p>
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Team"><font size = 4>ABOUT US</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Project"><font size = 4>PROJECT</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"<font size = 4>NOTEBOOK</font></span><br />
<a href="#meeting">Meeting Notes </a><br /><br />
<a href="#notebook">Lab Notebook</a><br /><br />
<a href="#protocols">Protocols</a><br/><br />
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Official Meetings <br />
</font></b></i> <hr><br />
<br />
<p class="header">May 12</p><br />
<br />
<p class = "class">Introduction to team members and to iGEM. Reviewed last year’s competition and last year’s team contribution. Introduction also to the 2009 iGEM home page and our team wiki. </p><br />
<br />
<p class="header">June 23</p><br />
<br />
<p class = "class">After researching previous projects and brainstorming since our last meeting, we discussed the possibility of continuing the project that University of Hawaii initiated last year. We spoke with an advisor from their team and agreed that we could continue the project. Our team agreed that it would be a good project foundation.</p> <br />
<br />
<p class="header">June 30</p><br />
<br />
<p class = "class">Small workshop on programming a wiki. Further collaboration with team Hawaii has taken place and we are waiting for DNA constructs with which they worked. </p><br />
<br />
<p class="header">August 20</p><br />
<br />
<p class = "class">Discussed progress made with broad-host vector construction. The broad-host vector constructs that we have been working with from Team Hawaii and from the iGEM parts catalog do not appear to be functioning. After PCR, attempted ligations, enzymatic digestions and electrophoretic gel observations, we’ve decided to move on and try to modify another known broad-host vector to be compatible with the BioBrick format. </p><br />
<br />
<p class="header">September 3</p><br />
<br />
<p class = "class">Team discussion of PHAs, phasin, silver-fusion, and secretory pathways. Further discussion of attempted broad-host vector modifications. </p><br />
<br />
<p class="header">September 10</p><br />
<br />
<p class = "class">Reviewed judging criteria and reviewed our standing with the broad-host vector and the secretion pathways. Discussed which tracks would be most applicable to our project. Discussed titles for our project. Finalization of team roster and travel information. </p><br />
<br />
<p class="header">September 17</p><br><br />
<br />
<p class = "class">Made final decisions for our intended track, chose a final project title, and gave a final review of our abstract. Discussed our wiki progress. </p><br />
<br />
<p class="header">September 24</p><br><br />
<br />
<p class = "class">Took team picture. Presentation of different team logo options and team shirt design options. Flash animation presentation to be used potentially in wiki and presentation. <br />
<br />
<p class="header">September 29</p><br><br />
<br />
<p class = "class">Team meeting with internet programming advisor. Discussed final formatting options for wiki. </p><br />
<br />
<p class="header">October 1</p><br><br />
<br />
<p class = "class">Instruction given on tri-parental mating. Discussed selective plates and media for tri-parental mating. </p><br />
<br />
<p class="header">October 6</p><br><br />
<br />
<p class = "class">Instruction given on Western Blot procedure and function. Discussed modified GFP construct. Final team logo was presented.</p><br />
<br />
<p class="header">October 8</p><br><br />
<br />
<br />
<p class = "class">Met shortly and separated to work on different assignments for wiki. </p><br />
<br />
<p class="header">October 13</p><br><br />
<br />
<p class = "class">Final T-shirt design presented to team. Reviewed completed parts and discussed broad-host vector. Presentation of completed secretion pathway models. </p><br />
<br />
<br />
<p class="header">October 15</p><br><br />
<br />
<br />
<p class = "class">Discussed portions of the wiki that needed to be completed before the weekend. Reviewed project components and iGEM basics in preparation for the jamboree. </p><br />
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<p class="header">October 20</p><br><br />
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<p class = "class">Final meeting before wiki closure. Discussed last minute assignments to ensure that the wiki is completed.</p><br />
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Laboratory Notebook<br />
</font></b></i> <hr><br />
<p class="class"><br />
Members of our team each had individual lab notebooks. Rather than outline each procedure that was run by each individual person, we have instead decided to present our wiki lab notebook as a weekly update up the progress that was made. Many of the various procedure details and specifics are found below in the protocols section. </p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/1/17/Notebooksusu.jpg" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Notebooks"> </div><br />
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<b>Figure 1.</b> Some of our laboratory notebooks<br />
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Protocols<br />
</font></b></i> <hr><br />
<p class="class"><br />
Members of our team each had individual lab notebooks. Rather than outline each procedure that was run by each individual person, we have instead decided to present our wiki lab notebook as a weekly update up the progress that was made. Many of the various procedure details and specifics are found below in the protocols section. </p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/1/17/Notebooksusu.jpg" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Notebooks"> </div><br />
<br><br />
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<b>Figure 1.</b> Some of our laboratory notebooks<br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State"><font size = 4>HOME</font></a></td><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>CONTACT</font></font></span><br />
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<b><i>Contact Us</b></i></font><br />
<HR><br />
<p class="class"><br />
We would like to hear your questions and comments! Please send us an email and come talk to us at the Jamboree! Thanks for your interest in USU iGEM 2009. Be sure to also check out the links to our sponsors web pages - we have greatly appreciated their help. </p><br />
<br />
<ul class="circle"><br />
<li>Dr. Charles Miller: charles.miller@engineering.usu.edu </li><br />
<li>The USU iGEM Team: iGEMUSU@gmail.com </li></ul><br />
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<td width="172" id="ana"><span class="currentPage"<font size = 4>NOTEBOOK</font></span><br />
<a href="#meeting">Meeting Notes </a><br /><br />
<a href="#notebook">Lab Notebook</a><br /><br />
<a href="#protocols">Protocols</a><br/><br />
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<b><i>Notebook<br />
</b></i></font><br />
<HR><br />
<br />
<p class = "class"> <br />
This page is for the notebook - this includes notes from our official team meetings, weekly progress updates, and the lab protocols.<br />
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<!---Official Meeting Notes---><br />
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Official Meetings <br />
</font></b></i> <hr><br />
<br />
<p class="header">May 12</p><br />
<br />
<p class = "class">Introduction to team members and to iGEM. Reviewed last year’s competition and last year’s team contribution. Introduction also to the 2009 iGEM home page and our team wiki. </p><br />
<br />
<p class="header">June 23</p><br />
<br />
<p class = "class">After researching previous projects and brainstorming since our last meeting, we discussed the possibility of continuing the project that University of Hawaii initiated last year. We spoke with an advisor from their team and agreed that we could continue the project. Our team agreed that it would be a good project foundation.</p> <br />
<br />
<p class="header">June 30</p><br />
<br />
<p class = "class">Small workshop on programming a wiki. Further collaboration with team Hawaii has taken place and we are waiting for DNA constructs with which they worked. </p><br />
<br />
<p class="header">August 20</p><br />
<br />
<p class = "class">Discussed progress made with broad-host vector construction. The broad-host vector constructs that we have been working with from Team Hawaii and from the iGEM parts catalog do not appear to be functioning. After PCR, attempted ligations, enzymatic digestions and electrophoretic gel observations, we’ve decided to move on and try to modify another known broad-host vector to be compatible with the BioBrick format. </p><br />
<br />
<p class="header">September 3</p><br />
<br />
<p class = "class">Team discussion of PHAs, phasin, silver-fusion, and secretory pathways. Further discussion of attempted broad-host vector modifications. </p><br />
<br />
<p class="header">September 10</p><br />
<br />
<p class = "class">Reviewed judging criteria and reviewed our standing with the broad-host vector and the secretion pathways. Discussed which tracks would be most applicable to our project. Discussed titles for our project. Finalization of team roster and travel information. </p><br />
<br />
<p class="header">September 17</p><br><br />
<br />
<p class = "class">Made final decisions for our intended track, chose a final project title, and gave a final review of our abstract. Discussed our wiki progress. </p><br />
<br />
<p class="header">September 24</p><br><br />
<br />
<p class = "class">Took team picture. Presentation of different team logo options and team shirt design options. Flash animation presentation to be used potentially in wiki and presentation. <br />
<br />
<p class="header">September 29</p><br><br />
<br />
<p class = "class">Team meeting with internet programming advisor. Discussed final formatting options for wiki. </p><br />
<br />
<p class="header">October 1</p><br><br />
<br />
<p class = "class">Instruction given on tri-parental mating. Discussed selective plates and media for tri-parental mating. </p><br />
<br />
<p class="header">October 6</p><br><br />
<br />
<p class = "class">Instruction given on Western Blot procedure and function. Discussed modified GFP construct. Final team logo was presented.</p><br />
<br />
<p class="header">October 8</p><br><br />
<br />
<br />
<p class = "class">Met shortly and separated to work on different assignments for wiki. </p><br />
<br />
<p class="header">October 13</p><br><br />
<br />
<p class = "class">Final T-shirt design presented to team. Reviewed completed parts and discussed broad-host vector. Presentation of completed secretion pathway models. </p><br />
<br />
<br />
<p class="header">October 15</p><br><br />
<br />
<br />
<p class = "class">Discussed portions of the wiki that needed to be completed before the weekend. Reviewed project components and iGEM basics in preparation for the jamboree. </p><br />
<br />
<p class="header">October 20</p><br><br />
<br />
<br />
<p class = "class">Final meeting before wiki closure. Discussed last minute assignments to ensure that the wiki is completed.</p><br />
<br />
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Laboratory Notebook<br />
</font></b></i> <hr><br />
<p class="class"><br />
Members of our team each had individual lab notebooks. Rather than outline each procedure that was run by each individual person, we have instead decided to present our wiki lab notebook as a weekly update up the progress that was made. Many of the various procedure details and specifics are found below in the protocols section. </p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/1/17/Notebooksusu.jpg" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Notebooks"> </div><br />
<br><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 1.</b> Some of our laboratory notebooks<br />
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<b><i><font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
Protocols<br />
</font></b></i> <hr><br />
<p class="class"><br />
Members of our team each had individual lab notebooks. Rather than outline each procedure that was run by each individual person, we have instead decided to present our wiki lab notebook as a weekly update up the progress that was made. Many of the various procedure details and specifics are found below in the protocols section. </p><br />
<br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/1/17/Notebooksusu.jpg" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Notebooks"> </div><br />
<br><br />
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<b>Figure 1.</b> Some of our laboratory notebooks<br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
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<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
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<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>BioBricks without Borders:<br />
</b></i></font><br />
<br><br />
<font size ="2.5" face="tahoma, sans-serif, verdana" color=#009900><br />
Investigating a multi-host BioBrick vector and secretion of cellular products</font><br />
<HR><br />
<br />
<p class = "class"> <br />
The aim of the Utah State University iGEM project is to develop improved upstream and downstream processing strategies for manufacturing cellular products using the standardized BioBrick system. First, we altered the broad-host range vector pRL1383a to comply with BioBrick standards and enable use of BioBrick constructs in organisms like Pseudomonas putida, Rhodobacter sphaeroides, and Synechocystis PCC6803. This vector will facilitate exploitation of advantageous characteristics of these organisms, such as photosynthetic carbon assimilation. Following expression, product recovery poses a difficult and expensive challenge. Downstream processing of cellular compounds, like polyhydroxyalkanoates (PHAs), commonly represents more than half of the total production expense. To counter this problem, secretion-promoting BioBrick devices were constructed through genetic fusion of signal peptides with protein-coding regions. To demonstrate this, the secretion of PHA granule-associated proteins and their affinity to PHA was investigated. Project success will facilitate expression and recovery of BioBrick-coded products in multiple organisms.<br />
</P> </td></tr><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
<br />
<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
<a href="#references">References</a><br />
</tr><br />
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<td><br />
<font size="5" face="Century Gothic, Arial, San Serif" color =#000033><br />
<b><i>BioBricks without Borders:<br />
</b></i></font><br />
<br><br />
<font size ="2.5" face="tahoma, sans-serif, verdana" color=#009900><br />
Investigating a multi-host BioBrick vector and secretion of cellular products</font><br />
<HR><br />
<br />
<p class = "class"> <br />
The aim of the Utah State University iGEM project is to develop improved upstream and downstream processing strategies for manufacturing cellular products using the standardized BioBrick system. First, we altered the broad-host range vector pRL1383a to comply with BioBrick standards and enable use of BioBrick constructs in organisms like Pseudomonas putida, Rhodobacter sphaeroides, and Synechocystis PCC6803. This vector will facilitate exploitation of advantageous characteristics of these organisms, such as photosynthetic carbon assimilation. Following expression, product recovery poses a difficult and expensive challenge. Downstream processing of cellular compounds, like polyhydroxyalkanoates (PHAs), commonly represents more than half of the total production expense. To counter this problem, secretion-promoting BioBrick devices were constructed through genetic fusion of signal peptides with protein-coding regions. To demonstrate this, the secretion of PHA granule-associated proteins and their affinity to PHA was investigated. Project success will facilitate expression and recovery of BioBrick-coded products in multiple organisms.<br />
</P> </td></tr><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/ExperimentsTeam:Utah State/Experiments2009-10-22T01:12:46Z<p>Liblint: </p>
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
<a href="https://2009.igem.org/Team:Utah_State/References">References</a><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Parts"><font size = 4>BIOBRICKS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Modeling"><font size = 4>MODELING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Notebook"><font size = 4>NOTEBOOK</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/ETHICS"><font size = 4>ETHICS</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Achievements"><font size = 4>JUDGING</font></a></td><br />
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<td id="nav"><a href="https://2009.igem.org/Team:Utah_State/Contact"><font size = 4>CONTACT</font></a></td><br />
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Experiments: Broad-Host Range Vectors<br />
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<p class = "class"> <br />
Converting Broad-Host Vectors into a BioBrick-Compatible Format<br />
<br />
Two Broad-host range vectors were used in this study; pRL1383a and PCPP33. To convert these vectors into BioBrick-compatible format, the four standard BioBrick sites EcoRI, XbaI, SpeI, and PstI needed to be inserted into the multiple cloning site. For pRL1383a, common BioBrick primers VR and VF2 were also included to allow the use of PCR in amplifying the BioBrick parts.<br />
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<br />
<br />
Experimental Section: Approach for BioBrick Compatibility<br />
<br />
Apart from being shown effective in the Synechosystis PCC 6803 (Marraccini 1993), pRL1383a is an ideal candidate for use as a BioBrick-compatible broad-host range vector because the BioBrick restriction sites are absent within the vector sequence. To convert pRL1383a into a BioBrick format, the existing multiple cloning site, which is flanked by a BamHI site and a HindIII site, was utilized. First, modified primers were synthesized from BioBrick primers VR and VF2. These primers were modified by adding extra nucleotides to insert the desired restriction enzyme sites into the PCR product. A BamHI site was added to 5’ end of the forward primer (VF2) and a HindIII site was added to the 5’ end of the reverse primer (VR). These primers were used to amplify an existing, tested BioBrick part by PCR. For this purpose, we selected BBa_I20260 because it does not contain BamHI or HindIII sites, and successful ligation is readily testable as it is a GFP -producing construct. The addition of IPTG is typically necessary to induce GFP production in this particular device. However, when using Top10 E. coli cells it is produced continuously because these cells lack a lac repressor (insert invitrogen link). After cutting the vector at the multiple cloning site using BamHI and HindIII, the BioBrick insert obtained by PCR with modified ends was ligated into the backbone. The vector was then transformed using Top10 One Shot® chemically competent E. coli and tested for successful insertion using PCR and restriction digests.<br />
<br />
Another broad host range vector, pCPP33, previously shown effective in Pseudomonas Putida,was standardized using similar methods. While the complete sequence of this plasmid is not available, it was shown that there are no BioBrick restriction sites outside the multiple cloning site (Figure 3). The multiple cloning site of this vector is flanked by EcoRI and HindIII. This allowed the PCR product of BBa_I20260 to again be used by cutting with HindIII and EcoRI restriction enzymes. Restriction digests and gel analysis were used to test for the insert.<br />
<br />
Broad Host Conjuation<br />
<br />
In order to transfer a vector of interest using conjugation, the tra gene (contained in what we will refer to as a transfer plasmid, or helper plasmid) must be expressed in order to initiate the conjugation process. This plasmid codes for genes which, when expressed, form pili on the cell surface, which in turn initiate conjugation (Heinemann 1989). This plasmid may be present in one of three different procedures:<br />
<br />
1) Hfr strain –The tra operon is many times contained in an episome, which can incorporate itself into the cell genome. These resultant Hfr strains will often begin the transfer of their own DNA, both plasmid and genomic. Due to the transfer of the genomic DNA, these strains are referred to as high frequency recombinant (Hfr) strains.<br />
<br />
2) Biparental (normal) Conjugation – Cells containing the tra genes, often labeled as F-positive (F+) due to the F-plasmid, a well-known transfer plasmid, can express the transfer genes necessary for conjugation to occur. When a vector of interest and a transfer plasmid are of different incompatibility groups, they may both be transformed into the same cell, and conjugation may take place between the F+ donor cell and the recipient cell<br />
<br />
3) Triparental Mating – In the case where the transfer plasmid and the vector of interest are of the same incompatibility group, the two plasmids may not stably coexist (Heinemann 1989). In this case, two separate cells containing the transfer gene (the helper cell) and the vector (the donor cell) must be used in conjugation. The helper cell will assist the donor cell in the transfer of its mobilizable plasmid to the recipient cell. This method circumvents some of the barriers that may prevent the transfer of plasmids.<br />
<br />
<br />
<br />
For our project, we chose to use the triparental mating procedure for the transmission of our vector. While not being the most efficient method, it circumvents possible barriers and intermediate steps.<br />
<br />
Because of the use of three different cells in our transformation procedure, the selection criteria for each component needed to be unique. In addition, we selected helper plasmids which had been known to work with the intended recipient cell.<br />
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<br />
<br />
Results<br />
<br />
Testing the ligation of pRL1383a and BBa_I20260 using PCR and restriction digests showed that the insert was not present in the vector, and the conversion to BioBrick format ultimately unsuccessful. The procedure as described above was repeated multiple times without success. Tri-parental conjugation of unmodified pRL 1383a was inconclusive in all target organisms.<br />
<br />
In an effort to troubleshoot this vector, several different approaches were taken. First, the ligation was repeated with varying concentrations of insert (10X, 2X) in an attempt to account for the impact of the large vector size on the ligation reaction. These ligations yielded similar results to reactions done at calculated concentrations. A Blunt-end ligation using a Klenow fragment was also performed. This was repeated, both attempts without success. The BBa_I20260 PCR product with BamHI/HindIII ends was ligated into another vector in an attempt to test the insert’s ability to be cut with the restriction enzymes. This ligation did not indicate the presence of the insert, suggesting that the problem lies with the vector or primers. The primers were tested and found viable on another insert, with similar testing of restriction enzymes to show functionality. The primers and enzymes were operating as intended, but new enzymes were ordered for more experimental certainty. The insert was then digested only with HindIII, and left in a ligation reaction. The outcome of this ligation was not of the desired length. This was repeated, and the same result obtained. While there is some suggestion that the BioBrick insert may not be functioning, the ambiguous results of tri-parental mating with unmodified pRL1383a suggests that the vector may be damaged or misunderstood.<br />
<br />
Testing the ligation of PCPP33 and BBa_I20260 also proved unsuccessful. Restriction digests using BioBrick standard pieces failed to yield an insert. Tri-parental mating of this vector proved successful in all organisms that we tested. All organisms yielded colonies on tetracycline plates, suggesting presence of the plasmid. Further testing by plasmid extraction and gel analysis will be done to conclusively determine presence of the plasmid. <br />
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Experiments: Secretion<br />
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Methods for Constructing BioBrick Parts<br />
</font><br></b><br />
<br />
<p class="class"><br />
One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length. <br />
<br />
<p class="class"><br />
Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.</p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Signal Peptides:</font></b><br><br />
<p class="class"><br />
To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.</p><br />
<p class="class"><br />
The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Phasin:</font></b><br><br />
<p class="class"><br />
The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis (LINK TO PROTOCOLS PAGE) (CTGCAG CTTCAG). Sequencing confirmed that this site was successfully removed. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>GFP:</font></b><br><br />
<p class="class"><br />
Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported. Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator. </p><br />
<br />
<p class="class"><br />
A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure X shows GFP- Top10 <i>E. coli</i> colonies (left) and unfused GFP+ Top10 <i>E. coli</i> colonies (right). This figure shows that the GFP construct is functional and easily detectable.</p><br><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/93/GFPglowingUSU.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Plate with GFP- cells (right) next to plate with GFP+ cells(left)<br />
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<br><b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Bioplastic Production:</font></b><br><br />
<p class="class">A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. <i>E. coli</i> harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure X. The observed peaks at 1.24 ppm, 2.54 ppm, and 5.2 ppm correspond with those observed in standard polyhydroxyalkanaote samples.</p><br />
<Br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/4/43/NMRusu.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Proton NMR spectra for PHB production in recombinant <i>E. coli</i><br />
</div><br />
<br><br />
<p class="class">To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>SDS-PAGE Analysis</font></b><br><br />
<p class="class">Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged (see Figure X). Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure X. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/d/d1/GFP_gel.png"" align = "middle" height="400" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing a strong band corresponding to GFP<br />
</div><br />
<br><br />
<p class="class">The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection, as shown in Figure X. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/3/3e/PHB_gel.png"" align = "middle" height="250" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing the presence of phasin protein in supernatant samples (third well from left)<br> next to supernatant from an <i>E. coli</i> sample without a phasin-producing construct.<br />
</div><br />
<Br><br />
<br />
<p class="class">Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Figure X shows the apparatus used to transfer proteins onto PVDF paper. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies. </p><br />
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Secretion">Secretion</a><br />
<a href="https://2009.igem.org/Team:Utah_State/Experiments">Experiments</a><br />
<a href="https://2009.igem.org/Team:Utah_State/FutureWork">Future Work</a><br />
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Experiments: Broad-Host Range Vectors<br />
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Experiments: Secretion<br />
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Methods for Constructing BioBrick Parts<br />
</font><br></b><br />
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<p class="class"><br />
One of the objectives of this project was to create a library of (link to silver fusion wiki) Silver-fusion compatible BioBrick signal peptides and protein-coding parts for secretion studies. The Silver-fusion assembly method was used because the standard BioBrick prefix and suffix do not facilitate fusion of two parts. The scar that forms from the overlap of compatible restriction enzyme sites XbaI and SpeI is not conducive to fusion because it contains a stop codon and is 8 nucleotides long. Because the scar is not a multiple of three, the sequence after the scar will be read out-of-frame. The Silver-fusion assembly method retains compatibility with the standard BioBrick assembly method, but fusion is allowed. A single nucleotide is removed from the prefix and suffix of Silver-fusion BioBricks so that the scar that forms from the ligation of XbaI and SpeI sites does not contain a stop codon and is 6 nucleotides in length. <br />
<br />
<p class="class"><br />
Five signal sequences were selected for this study based on the secretion pathway that they represent and their prominence in literature. The selected sequences are presented in Table X. Two protein coding regions were obtained: phasin and GFP. All of these sequences were designed for Silver-fusion compatibility. Four different promoters with an attached ribosome binding site were designed and then synthesized by DNA 2.0, followed by ligation into a BioBrick vector. Composite devices were assembled piecewise by cutting one part typically with EcoRI and XbaI, and the part to be inserted with EcoRI and SpeI. Analysis by PCR with the Primers VF2 and VR was used to qualitatively determine whether successful ligation had taken place. Once partially confirmed, samples were sequence at the Utah State University Center of Integrated Biotechnology.</p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Signal Peptides:</font></b><br><br />
<p class="class"><br />
To construct the OmpA, PelB, and GeneIII sequences, complimentary forward and reverse oligonucleotides were synthesized by Eurofins Operon. These strands were then annealed together. The oligonucleotides were designed so that the silver fusion prefix and suffix sequences were appended onto the end of each sequence. These parts were then cut with EcoRI and SpeI and ligated into a BioBrick vector. Each of these parts were successfully constructed and sequenced.</p><br />
<p class="class"><br />
The TorA and HlyA signal peptides were synthesized by DNA 2.0 because these sequences are longer than the other signal peptides, which made the complimentary oligonucleotides method not ideal. The Silver-fusion prefix and suffix was added to each of these constructs. EcoRI and SpeI were used to cut the part out of the commercial vector. The DNA was isolated by gel electrophoresis and ligated into a BioBrick compatible vector, pSB3K3. </p><br />
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<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Phasin:</font></b><br><br />
<p class="class"><br />
The phasin (PhaP) sequence was isolated from the genomic DNA of Cupriavidus necator (also known as Ralstonia eutropha). There are four different phasin genes in the genomic DNA of this organism. This particular phasin was selected based on references in literature, although no information was acquired that indicated that one phasin gene would yield better production over another. The primers were designed so that the Silver-fusion prefix and suffix were overhanging, thereby resulting in a final product that is Silver-fusion compatible. The 579 bp phasin sequence was found to contain a PstI site. The PstI site was mutated using site-directed mutagenesis (LINK TO PROTOCOLS PAGE) (CTGCAG CTTCAG). Sequencing confirmed that this site was successfully removed. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>GFP:</font></b><br><br />
<p class="class"><br />
Near the beginning of this project, a Silver-fusion compatible GFP BioBrick (BBa_K125500) derived from BBa_E0040 by the Hawaii 2008 iGEM team was obtained. However, upon further analysis it was determined that this part was modified so that the start codon of the sequence was removed. Although this should not affect the expression of GFP in composite parts with a signal peptide prior to the sequence, it is not ideal for this particular project. The lack of a start codon requires N-terminal fusion of another protein or signal peptide, and a functional GFP control without a signal sequence would not be functional. This control is important in our study to compare with composite parts containing signal peptide-protein fusion to determine whether the produced GFP is being transported. Additionally, this part would not work with C-terminal signal peptide fusions. The HlyA signal peptide is recognized on the C-terminus of the target protein by the Type I secretion pathway (Mergulhão, 2005; Gentschev, 2003; Hess, 1990). The absence of the start codon inhibits study of this secretory pathway. Another disadvantage of this GFP part is its small Stokes shift (excitation 501 nm, emission 511 nm). An ideal GFP that fluorescence would have a shorter excitation wavelength so that GFP-positive samples can be detected visually using a UV transilluminator. </p><br />
<br />
<p class="class"><br />
A new Silver-Fusion compatible GFP BioBrick part was constructed for this project via a similar mechanism as the phasin construct. This particular GFP was previously mutated for improved fluorescence photostability (Crameri, 1996). The excitation and emission wavelengths for this GFP are 395 nm and 501 nm, respectively. That being said, GFP-positive cells emit a bright green fluorescence when exposed to shorter-wavelength UV light, such as on a transilluminator. Primers were synthesized for isolation of the sequence and, like the phasin-specific primers, designed so that the Silver-fusion prefix and suffix were inserted on the ends of the sequence (see primers). Figure X shows GFP- Top10 <i>E. coli</i> colonies (left) and unfused GFP+ Top10 <i>E. coli</i> colonies (right). This figure shows that the GFP construct is functional and easily detectable.</p><br><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/9/93/GFPglowingUSU.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Plate with GFP- cells (right) next to plate with GFP+ cells(left)<br />
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<br><b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>Bioplastic Production:</font></b><br><br />
<p class="class">A plasmid harboring the genes for PHB production (pBHR68) was used in these experiments. This plasmid contains the sequence for ampicillin resistance and contains a ColE1 origin of replication. <i>E. coli</i> harboring pBHR68 were cultured according to methods outlined by Kang et al (2008) and production of PHB was verified using 1H NMR analysis. The spectrum obtained from this experiment is given as Figure X. The observed peaks at 1.24 ppm, 2.54 ppm, and 5.2 ppm correspond with those observed in standard polyhydroxyalkanaote samples.</p><br />
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<div align="center"><img src="https://static.igem.org/mediawiki/2009/4/43/NMRusu.jpg"" align = "middle" height="200" style="padding:.5px; border-style:solid; border-color:#999" alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Proton NMR spectra for PHB production in recombinant <i>E. coli</i><br />
</div><br />
<br><br />
<p class="class">To maintain plasmid compatibility in E. coli transformed with both the pBHR68 and phasin plasmids, it was determined that the vector used for the phasin secretion device required a p15A ori site. BioBrick vector pSB3K3 was found suitable as the host for the secretion constructs. XL1-Blue E. coli were transformed with both a phasin device and the pBHR68 BioBrick plasmids, and these cells were cultured and tested for secretion. </p><br />
<br />
<b><font size="2.5" face="Arial, Helvetica, San Serif" color =#231f20>SDS-PAGE Analysis</font></b><br><br />
<p class="class">Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the protein content in transformed E. coli. As a positive control, E. coli containing the Lac/RBS/GFP/Terminator (BBa_K208045) construct were sonicated and centrifuged (see Figure X). Additionally, E. coli cells containing an individual BioBrick part (BBa_B0015) were analyzed as a negative control. The resulting gel was stained with coomassie blue and is shown as Figure X. The bright band at 27 kD in the GFP+ sample corresponds to the GFP protein (Bio-Rad). The absence of this band in the GFP- sample further reinforces the functionality of the GFP construct.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/d/d1/GFP_gel.png"" align = "middle" height="400" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing a strong band corresponding to GFP<br />
</div><br />
<br><br />
<p class="class">The geneIII secretion signal sequence fused to the phasin protein was expressed in E. coli cells. The E. coli cells were grown overnight in LB growth media and centrifuged to pellet the cells. Supernatants (5ml) were then concentrated using a Centricon Centriplus concentrator (Amicon, Beverly MA). This process concentrated proteins that were larger than 10kDa and removed molecules smaller than 10kDa. Approximately 20ug of protein were then applied to a SDS polyacrylamide gel to separate the proteins according to size. The gel was then stained with coomassie blue for protein detection, as shown in Figure X. Following SDS polyacylamide gel electrophoresis (PAGE) and subsequent coomassie blue staining of the separated proteins, a protein with an approximate size of 22kDA is observed in the sample from the phasin-expressing E. coli cells that is not present in the control E. coli sample. The phasin protein has been reported by others to migrate on SDS PAGE from 14-28kDa (Pötter, 2002; York, 2002). These results indicate that the GeneIII::phasin expression construct is being produced by the E. coli cells and is being secreted outside the cell into the media.</p><br />
<br><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2009/3/3e/PHB_gel.png"" align = "middle" height="250" style="padding:.5px; alt="Team USU" /> </div><br />
<div align="center"><font size="2.5" face="Helvetica, Arial, San Serif" color =#231f20><br />
<b>Figure 2.</b> Protein gel showing the presence of phasin protein in supernatant samples (third well from left)<br> next to supernatant from an <i>E. coli</i> sample without a phasin-producing construct.<br />
</div><br />
<Br><br />
<br />
<p class="class">Western blotting with phasin-specific antibodies was performed to verify the observed band as phasin. Figure X shows the apparatus used to transfer proteins onto PVDF paper. Phasin antibody was kindly provided by Anthony J. Sinskey at Massachusetts Institute of Technology. The results of the western blotting were inconclusive. Non-specific binding to larger constructs was observed. Additional testing is required to further reinforce preliminary findings and confirm the secretion of phasin. The secretion of phasin would provide evidence that PHA recovery via phasin secretion is possible. Addtionally, this would reinforce that the constructed BioBricks are not only functional, but would be beneficial for use in other studies. </p><br />
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</head></div>Liblinthttp://2009.igem.org/Team:Utah_State/ReferencesTeam:Utah State/References2009-10-22T01:09:35Z<p>Liblint: New page: <!--- BANNER---> <html> <a href="https://2009.igem.org/Team:Utah_State"> <img alt="USU iGem" src="https://static.igem.org/mediawiki/2009/7/71/USUlogo.jpg"/> </a> <!--- Table---> <!DOCTYPE html ...</p>
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<td width="172" id="ana"><span class="currentPage"><font size = 4>PROJECT</font></span><br />
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<a href="https://2009.igem.org/Team:Utah_State/Abstract">Abstract</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Introduction">Introduction</a><br /><br />
<a href="https://2009.igem.org/Team:Utah_State/Broad-HostVectors">Broad-Host Vectors</a><br /><br />
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