Team:Minnesota/Notebook

From 2009.igem.org

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{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]
!align="center"|[[Team:Minnesota|<font color="gold">Home</font>]]
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]
!align="center"|[[Team:Minnesota/Team|<font color="gold">The Team</font>]]
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]
!align="center"|[[Team:Minnesota/Project|<font color="gold">The Project</font>]]
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!align="center"|[[Team:Minnesota/Parts|<font color="gold">Submitted Parts</font>]]
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!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]
!align="center"|[[Team:Minnesota/Modeling|<font color="gold">Modeling</font>]]
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!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]
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!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experimental</font>]]
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!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Parts Characterization</font>]]
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!align="center"|[[Team:Minnesota/Parts Characterization|<font color="gold">Competition Requirements</font>]]
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!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experiments and Calendar</font>]]
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<html>
+
<h2>Overview</h2>
-
<body>
+
This year’s AND gate project is a continuation of previous years.  In the past different combinations of Tet, Lac, and null operator sites were made to produce an AND gate.  From last year results, we decided to use the Tet Tet Lac promoter because it was the least leaky.  We decided to mutate the palindromic sequence in the Tet operator site.  This would alter the binding affinity of TetR for the operator site.  By doing this, we can have the modeling team more accurately predict the behavior of our promoter.  We also made and tested the TNN and TTN constructs in the hopes that the models would add together thermodynamically to be equivalent to our AND gate data.
-
<h1>The Experiments</h1>
+
 +
<h2>Standard Protocols We Used in the Wet Lab</h2>
 +
<h5>These link to pdf files:</h5>
 +
[[Media:Bacterial_Culture.pdf|Bacterial Culture Protocols]]
-
<h1>Results</h1>
+
[[Media:Transformation_of_Chemically_Competent_Cells.pdf|Transformation of Chemically Competent Cells]]
 +
[[Media:Plasmid_Prep_from_Cultures.pdf|Plasmid Prep from Cultures]]
 +
[[Media:DNA_Quantification.pdf|DNA Quantification]]
-
<h1>Protocols: Standard techniques that we used in the wet lab</h1>
+
[[Media:PCR.pdf|Polymerase Chain Reaction (PCR)]]
-
<h3>Bacterial Culture</h3>
+
 
-
<h4>Sterile Technique</h4>
+
[[Media:Restriction_Digest.pdf|Restriction Digest]]
-
<ol>
+
 
-
<li>Always work around a flame or in the hood</li>
+
[[Media:Vector_Dephosphorylation.pdf|Vector Dephosphorylation]]
-
<li>Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping</li>
+
 
-
<li>Sterilize metal instruments between uses by dipping in 100% ethanol and flaming</li>
+
[[Media:DNA_Fragment_Ligation.pdf|DNA Fragment Ligation]]
-
</ol>
+
 
 +
[[Media:DNA_Purification.pdf|DNA Purification]]
 +
 
 +
[[Media:Sequencing.pdf|Sequencing]]
 +
 
 +
[[Media:Preparing_Competent_Cells.pdf|Preparing Competent Cells]]
 +
 
 +
[[Media:Soeing_PCR.pdf|SOEing PCR]]
 +
 
 +
[[Media:Ligation_Reaction.pdf|Ligation]]
 +
 
 +
[[Media:Screening.pdf|Screening]]
 +
 
 +
[[Media:Sample_Collection.pdf|Sample Collection]]
<br />
<br />
-
<h4>Bacterial Culture Maintenance</h4>
+
<h2>Procedure</h2>
-
Culture cells:
+
 
<ol>
<ol>
-
<li>At 36 degrees Celsius</li>
+
<li>[http://2009.igem.org/wiki/images/4/4a/Preparing_Competent_Cells.pdf Prepare Competent Cells] for TOP10 and DH5αPro</li>
-
<li>Shaking at 220 rpm</li>
+
<li>Design the primers so that the proper mutations exist in the palindromic sequence of the Tet operator site.  Design for each construct: TNN, TTN, and TTL.</li>
-
<li>At 10% total flask/tube volume</li>
+
<li>Combine [http://2009.igem.org/wiki/images/a/ae/Soeing_PCR.pdf Soeing PCR] reagents with primers and place in a thermocycler.</li>
-
<li>In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x10<sup>8</sup>cell/ml)
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<li>Amplify the insert using [http://2009.igem.org/wiki/images/7/74/PCR.pdf PCR]</li>
 +
<li>Purify the PCR product using [http://2009.igem.org/wiki/images/a/a8/DNA_Purification.pdf QIAquick PCR purification]</li>
 +
<li>Ligate[http://2009.igem.org/wiki/images/b/b3/Ligation_Reaction.pdf Ligation] the insert into pGLOTopo</li>
 +
<li>The plasmid is [http://2009.igem.org/wiki/images/f/f5/Transformation_of_Chemically_Competent_Cells.pdf transformed] into TOP10 cells</li>
 +
<li>The transformants are plated then screened [http://2009.igem.org/wiki/images/6/66/Screening.pdf screened] using a florescent camera</li>
 +
<li>Once the colonies have been screened, positive colonies have their plasmids isolated[http://2009.igem.org/wiki/images/3/39/Plasmid_Prep_from_Cultures.pdf plasmids isolated].</li>
 +
<li>The plasmids are then [http://2009.igem.org/wiki/images/b/b0/Sequencing.pdf sequenced] to ensure the correct sequence was obtained.</li>
 +
<li>Transform the cells into DH5αPro and TOP10 and perform [http://2009.igem.org/wiki/images/3/32/Sample_Collection.pdf sample collection]</li>
 +
<li>The samples are left at 4 °C for 24 hours.</li>
 +
<li>The relative GFP intensity is taken with FACSCalibar flow cytometer.</li>
</ol>
</ol>
 +
<br />
<br />
-
<h4>Bacterial Culture For Gene Expression Experiments</h4>
+
<h2>Discussion of General Trends Found</h2>
-
<ol>
+
 
-
<li>Pick and individual colony from a plate and inoculate 2ml LB + amp media</li>
+
The positive control showing a weak signal can be explained by the GFP forming inclusion bodies in high concentrations.  The inclusion bodies would be fluorescently inactive and show a negative GFP signal.  This theory is also support by Figure 1.C which shows two peaks for two time points.  At the earlier time point, the negative GFP peak is weak while the positive gives a strong signal, however at the later time point the negative GFP signal is stronger and the positive GFP.  There is also roughly the same area under both peaks meaning that it is likely that the counts from one event are being transferred to the other. Since time is the only factor and GFP accumulates over time, it can be inferred that at high concentration the cell forms the fluorescently inactive inclusion bodies.   The large increase from an aTc concentration of 10 ng to 50 ng that is observed is likely explained by the saturation of TetR.  With the TetR bound to its inducers, it wouldn’t bind to the operator site and produce a greater GFP intensity.  However, the rate of GFP production in the TTL construct seems to be governed more strongly by the Lac operator site.  This is likely due to LacR being a very large molecule which prevents polymerase from binding to the operator site.
-
<li>Incubate overnight at 37 C, shaking at 220 rpm</li>
+
The mutations in the Tet operator site were made to change the binding affinity of TetR for the operator site.  These alterations not only affect the binding affinity of just the repressor protein but also the inducer-repressor protein complex.  This means that not only the leakiness of the promoter will be affected.  The efficiency of repression is also affected.  Our results from the AND gate expression did not support what was found when just studying just mutations in the Tet operator .  The highest expression was observed with mutant 4 while mutant 3 and 5 had a slight decrease in efficiency of transcription.
-
<li>Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture</li>
+
 
-
<li>Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)</li>
+
 
-
<li>Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture</li>
+
 
-
<li>Continue cultures as described above in "bacterial culture maintenance" for 9 hrs</li>
+
[[Image:IGem_4.jpg|350px|left|TTL T0P10]][[Image:IGem_2.jpg|350px|right]]
-
<li>Isolate cell samples from cultures at 3, 6, and 9 hour time points</li>
+
 
-
<li>Remove 100ul sample aliquots from cultures</li>
+
 
-
<li>Pellet samples at 5K rpm for 5 minutes</li>
+
 
-
<li>Remove supernatant</li>
+
 
-
<li>Wash cells with 1 ml chilled 1xPBS, pH 7.6</li>
+
 
-
<li>Resuspend cells by vortexing</li>
+
 
-
<li>Re-pellet cells at 5K rpm for 5 minutes</li>
+
 
-
<li>Remove supernatant</li>
+
 
-
<li> Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)</li>
+
[[Image:IGem_1.jpg|350px|center]]
-
<li>Incubate at RT for 30 minutes</li>
+
 
-
<li>Pellet cells at 5K rpm for 5 minutes</li>
+
 
-
<li>Remove supernatant</li>
+
 
-
<li>Resuspend cells in 1 ml 1xPBS</li>
+
 
-
<li> Store samples at 4 C until analysis by flow cytometry</li>
+
 
-
</ol>
+
 
 +
<h2>Safety</h2>
 +
1. Would any of your project ideas raise safety issues in terms of:
 +
researcher safety, public safety, or environmental safety?
<br />
<br />
-
<h4>Transformation of Chemically Competent Cells</h4>
+
No, our constructs are simple promoter regulatory elements, built from well characterized lactose and tetracycline operon components, and characterized in non-pathogenic strain of E. coli.  
-
<ol>
+
-
<li>Thaw cells and incubate transformant DNA in ice(~15 minutes)</li>
+
-
<li>Combine 50 ul cells with ~3uL DNA and mix gently</li>
+
-
<li>Incubate samples on ice for 15 minutes</li>
+
-
<li>Heat shock cells in 42C water bath of 50 seconds</li>
+
-
<li>Incubate samples on ice for 5 minutes</li>
+
-
<li>Recover cells in 0.5 ml SOC media, shaking at 37C for 1 hour at 220 rpm</li>
+
-
<li>Transfer cells to a 2 ml microfuge tube</li>
+
-
<li>Spin cells down at 6K rpm for 2 minutes</li>
+
-
<li>Remove all but ~100uL supernatant media</li>
+
-
<li>Resuspend cells gently in remaining media</li>
+
-
<li>Plate cells on LB + ab plates</li>
+
-
<li>Incubate plates overnight at 37C</li>
+
-
</ol>
+
<br />
<br />
 +
2. Is there a local biosafety group, committee, or review board at your institution?
<br />
<br />
 +
Yes, there is an Institutional Biosafety Committee at the University of Minnesota. We have described our work and gotten permission to work in a wet lab (IBC Code Number: 0706H11321).
<br />
<br />
-
<h3>DNA Work</h3>
+
3. What does your local biosafety group think about your project?
<br />
<br />
-
<h4>Plasmid Prep from cultures (using QIAprep Spin Miniprep Kit)</h4>
+
We use standard molecular biology techniques. IBC readily approved a continuing review.
-
<ol>
+
-
<li>Pick and individual colony from a plate and inoculate 2ml LB + ab media</li>
+
-
<li>Incubate culture overnight at 37C</li>
+
-
<li>Transfer culture to 2 ml microfuge tube</li>
+
-
<li>Spin cells down at 13K rpm for 2 min at RT and remove supernatant</li>
+
-
<li>Resuspend cells in 250 ul Buffer P1 (stored at 4 C)</li>
+
-
<li>Add 250 ul Buffer P2 and mix thoroughly by inverting-- the solution should turn blue</li>
+
-
<li>Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting-- the solution should turn colorless</li>
+
-
<li>Centrifuge sample at 13K rpm for 10 minutes</li>
+
-
<li>Transfer supernatant to a fresh QIAprep spin column, leaving cell debris pellet behind</li>
+
-
<li>Centrifugre supernatant into column at 13K rpm for 1 minute</li>
+
-
<li>Remove the flowthrough</li>
+
-
<li>Wash column with 0.5 ml Buffer PB; apply to column and spin through at 13K rpm for 1 minute</li>
+
-
<li>Remove the flowthrough</li>
+
-
<li>Wash column with 0.75 ml Buffer PE; apply to column and spin through at 13K rpm for 1 minute</li>
+
-
<li>Remove the flowthrough</li>
+
-
<li>Spin out residual liquid at 13K rpm for 1 minute</li>
+
-
<li>Place column in a fresh 1.5 ml microfuge tube</li>
+
-
<li>Elute DNA; apply 40 ul Buffer EB to column, incubate at room temperature for 2 minutes and spin out of column at 13K rpm for one minute</li>
+
-
</ol>
+
<br />
<br />
-
<h4>DNA quantification</h4>
+
4. Do any of the new BioBrick parts that you made this year raise any safety issues? If yes, did you document these issues in the Registry?
-
<ol>
+
-
<li>Dilute DNA as appropriate in water (1<= DF <=1/100) to a total volume of 50 ul</li>
+
-
<li>Similarly dilute blank DNA buffer solution with water to a total volume of 50 ul</li>
+
-
<li>Read absobance of blank and DNA sample at lambda = 260 and 280</li>
+
-
<li>Calculate [DNA]; [DNA](ng/ul) = DF*A260*50</li>
+
-
<li>Determine sample purity; pure DNA A260/A280 = 1.8</li>
+
-
</ol>
+
<br />
<br />
-
<h4>Polymerase Chain Reaction(PCR)</h4>
+
No, none of the parts we built raise any safety issues.
-
<ol>
+
 
-
<li>Combine the following on ice:</li>
+
-
<table>
+
-
<table border="1">
+
-
<tr>
+
-
<th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
+
-
</tr>
+
-
<tr>
+
-
<td>10x Thermo Pol Buffer</td><td>5</td><td>1x</td>
+
-
</tr>
+
-
<tr>
+
-
<td>10mM dNTPs</td><td>1</td><td>0.2mM</td>
+
-
</tr>
+
-
<tr>
+
-
<td>50mM MgCl<sub>2</sub></td><td>2</td><td>2mM</td>
+
-
</tr>
+
-
<tr>
+
-
<td>10 uM F Primer</td><td>2</td><td>0.4mM</td>
+
-
</tr>
+
-
<tr>
+
-
<td>10 uM R Primer</td><td>2</td><td>0.4uM</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td>H<sub>2</sub>O</td><td>Vol req for 50 ul total</td><td>-</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td>DNA</td><td>Vol req for 30 ng <= mass <= 500ng</td><td>-</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td>Taq polymerase</td><td>0.5</td><td>2 U</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td><b>Total</b></td><td><b>50 ul</b></td><td>-</td><td>
+
-
</tr>
+
-
</table>
+
-
<li>Thermocycle</li>
+
-
<table>
+
-
<table border="1">
+
-
<tr>
+
-
<th>Segment</th><th>Temperature(C)</th><th>Length<th>
+
-
</tr>
+
-
<tr>
+
-
<td>1. Initial Denaturation</td><td>94</td><td>3 minutes</td>
+
-
</tr>
+
-
<tr>
+
-
<td>2. Denaturation</td><td>94</td><td>30 seconds</td>
+
-
</tr>
+
-
<tr>
+
-
<td>3. Annealing</td><td>~55</td><td>30 seconds</td>
+
-
</tr>
+
-
<tr>
+
-
<td>4. Extension</td><td>68</td><td>1 min/kb amplified</td>
+
-
</tr>
+
-
<tr>
+
-
<td>5. Final Extension</td><td>68</td><td>5 minutes</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td>6. Final Hold</td><td>12</td><td>Forever</td><td>
+
-
</tr>
+
-
</table>
+
-
<li>Repeat 25-35 cycles of segments 2-4</li>
+
-
</ol>
+
<br />
<br />
-
<h4>Restriction Digest</h4>
+
<html>
-
<ol>
+
<body>
-
<li>Combine the following on ice:</li>
+
 
-
<table>
+
<h2>Notebook</h2>
-
<table border="1">
+
This calendar contains a day-by-day catalog of what we did in the wet lab for our project and parts characterization. The calendar for computational work can be found below and on the
-
<tr>
+
<a href="http://2009.igem.org/Team:Minnesota/Modeling">Modeling</a> page.
-
<th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
+
Please click on each event to see a detailed description of what we did.  
-
</tr>
+
-
<tr>
+
-
<td>10x NEB Buffer</td><td>5</td><td>1x</td>
+
-
</tr>
+
-
<tr>
+
-
<td>BSA</td><td>0.5</td><td>1x</td>
+
-
</tr>
+
-
<tr>
+
-
<td>H<sub>2</sub>O</td><td>Vol req for 50 ul total</td><td>-</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td>DNA</td><td>Vol req for 0.5 ng <= mass <= 5 ug</td><td>-</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td>Restriction Enzyme</td><td>0.5</td><td>1 U</td><td>
+
-
</tr>
+
-
<tr>
+
-
<td><b>Total</b></td><td><b>50 ul</b></td><td>-</td><td>
+
-
</tr>
+
-
</table>
+
-
<li>Incubate at 37C for 2-20 hours</li>
+
-
<li>Heat inactive the enzyme at 65C for 15 minutes</li>
+
-
</ol>
+
<br />
<br />
-
<h4>Vector Dephosphorylation</h4>
 
-
<ol>
 
-
<li>Combine the following on ice</li>
 
-
<table>
 
-
<table border="1">
 
-
<tr>
 
-
<th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
 
-
</tr>
 
-
<tr>
 
-
<td>10x AP Buffer</td><td>5</td><td>1x</td>
 
-
</tr>
 
-
<tr>
 
-
<td>H<sub>2</sub>O</td><td>Vol req for 50 ul</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA</td><td>Vol req for 0.5 ug <= mass <= 5ug</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Antarctic Phosphate</td><td>0.5</td><td>1 U</td>
 
-
</tr>
 
-
</table>
 
-
<li>Incubate at 37C for 30 minutes</li>
 
-
<li>Heat inactive enzyme at 65C for 15 minutes</li>
 
-
</ol>
 
-
<br />
 
-
<h4>DNA Fragment Ligation</h4>
 
-
<ol>
 
-
<li>Combine the following on ice:</li>
 
-
<table>
 
-
<table border="1">
 
-
<tr>
 
-
<th>Reagent</th><th>1x(volume in ul)</th><th>[final]<th>
 
-
</tr>
 
-
<tr>
 
-
<td>10x Ligase Buffer</td><td>2</td><td>1x</td>
 
-
</tr>
 
-
<tr>
 
-
<td>H<sub>2</sub>O</td><td>Vol req for 20 ul</td><td>-</td>
 
-
<tr>
 
-
<td>Vector DNA</td><td>3 <= fmoles <= 30</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Insert DNA</td><td>9 <= fmoles <=90</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>T4 DNA Ligase</td><td>1</td><td>1 U</td>
 
-
</tr>
 
-
<tr>
 
-
<td><b>Total</b></td><td><b>20</b></td><td>-</td>
 
-
</tr>
 
-
</table>
 
-
<li>Vector insert ratio should be about 1:3-- lower ratios may decrease insertion efficiency and higher ratios may lead to cancatamerization of inserts. Additionally, 100ng <= total DNA <= 500ng</li>
 
-
<li>Incubate at 16C overnight or at RT for 30 minutes(overnight ligation is preferred)</li>
 
-
<li>Heat inactivate enzyme at 65C for 15 minutes</li>
 
-
</ol>
 
-
<br />
 
-
<h4>DNA purification</h4>
 
-
<h5>Purify DNA (Using QIAquick PCR purification)</h5>
 
-
<ol>
 
-
<li>Add 5 volumes of Buffer PB1to 1 volume DNA and mix</li>
 
-
<li>Apply DNA sample to QIA quick column</li>
 
-
<li>Spin DNA into column at 13K rpm for 1 minute</li>
 
-
<li> Discard flowthrough</li>
 
-
<li>Wash DNA with 0.75 ml Buffer PE, spin through column at 13K rpm for 1 minute</li>
 
-
<li>Discard flowthrough</li>
 
-
<li>Spin residual liquid from column at 13K rpm for 1 minutes</li>
 
-
<li>Elute DNA; apply 40ul Buffer EB to column, incubate at room temperature for 2 minutes before spinning DNA out of column at 13K rpm for 1 minute</li>
 
-
</ol>
 
-
<h5>Separate DNA by size on an agarose gel</h5>
 
-
<ol>
 
-
<li>Make an agarose gel at 0.8<= gel density <= 1.5</li>
 
-
<li> Add loading dye to samples (5 ul dye/50 ul sample)</li>
 
-
<li>Load samples and a ladder (5 ul) into gel wells</li>
 
-
<li>Run samples through gel (negative to positive) at 100 V for 40 minutes at room temperature</li>
 
-
<li>Visualize DNA under UV light</li>
 
-
</ol>
 
-
<h5>Purify DNA from Gel (Using QIAquick Gel Extraction Kit</h5>
 
-
<ol>
 
-
<li>Excise gel piece containing DNA with a new razor</li>
 
-
<li>Add three volumes of Buffer QG to 1 volume gel</li>
 
-
<li>Incubate at 50C for 15 minutes, or until gel is solublized, mixing frequently</li>
 
-
<li>Make sure dissolved solution is yellow</li>
 
-
<li>If the DNA fragment is <500bp and >4kb, add 1 gel volume isopropanol to increase the yield</li>
 
-
<li>Apply sample to QIAquick spin column in 700 ul aliquots</li>
 
-
<li>Spin sample into column at 13K rpm for 1 minute</li>
 
-
<li>Discard flowthrough</li>
 
-
<li>Spin 0.5 ml Buffer QG through column at 13K rpm for 1 minute to solublize any remaining gel chunks</li>
 
-
<li>Discard flowthrough</li>
 
-
<li>Wash column with 0.75 ml Buffer PE, spinning through column at 13K rpm for 1 minute</li>
 
-
<li>Discard flowthrough</li>
 
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<li>Spin out residual liquid from column at 13K rpm for 1 minute</li>
 
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<li>Elute DNA by applying 40-50ul Buffer EB to column, incubate at room temperature for 2 minutes, spin DNA out of the column at 13K rpm for 1 minute into a clean microfuge tube</li>
 
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</ol>
 
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<br />
 
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<h3>Sequencing</h3>
 
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<ol>
 
-
<li>Combine the following in 0.5ml epindorf tube:</li>
 
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<table>
 
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<table border="1">
 
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<th>Reagent</th><th>1x(vol in ul)</th><th>[Final]<th>
 
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<td>10 uM seq primer</td><td>1</td><td>10 pmol</td>
 
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</tr>
 
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<tr>
 
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<td>DNA</td><td>Vol req for 300ng</td><td>300 ng</td>
 
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</tr>
 
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<tr>
 
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<td>H<sub>2</sub>O</td><td>Vol req for 12 uL</td><td>-</td>
 
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</tr>
 
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<tr>
 
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<td><b<Total Volume</b></td><td><b>12 ul</b></td><td>-</td>
 
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</tr>
 
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</table>
 
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<li>Submit to BMGC for sequencing</li>
 
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</ol>
 
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<h2>Our Google Calendar</h2>
 
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Latest revision as of 21:31, 23 June 2010

Mnlogo.jpg
Home The Team The Project SynBioSS Designer Modeling Experimental Competition Requirements

Contents

Overview

This year’s AND gate project is a continuation of previous years. In the past different combinations of Tet, Lac, and null operator sites were made to produce an AND gate. From last year results, we decided to use the Tet Tet Lac promoter because it was the least leaky. We decided to mutate the palindromic sequence in the Tet operator site. This would alter the binding affinity of TetR for the operator site. By doing this, we can have the modeling team more accurately predict the behavior of our promoter. We also made and tested the TNN and TTN constructs in the hopes that the models would add together thermodynamically to be equivalent to our AND gate data.

Standard Protocols We Used in the Wet Lab

These link to pdf files:

Bacterial Culture Protocols

Transformation of Chemically Competent Cells

Plasmid Prep from Cultures

DNA Quantification

Polymerase Chain Reaction (PCR)

Restriction Digest

Vector Dephosphorylation

DNA Fragment Ligation

DNA Purification

Sequencing

Preparing Competent Cells

SOEing PCR

Ligation

Screening

Sample Collection

Procedure

  1. Prepare Competent Cells for TOP10 and DH5αPro
  2. Design the primers so that the proper mutations exist in the palindromic sequence of the Tet operator site. Design for each construct: TNN, TTN, and TTL.
  3. Combine Soeing PCR reagents with primers and place in a thermocycler.
  4. Amplify the insert using PCR
  5. Purify the PCR product using QIAquick PCR purification
  6. LigateLigation the insert into pGLOTopo
  7. The plasmid is transformed into TOP10 cells
  8. The transformants are plated then screened screened using a florescent camera
  9. Once the colonies have been screened, positive colonies have their plasmids isolatedplasmids isolated.
  10. The plasmids are then sequenced to ensure the correct sequence was obtained.
  11. Transform the cells into DH5αPro and TOP10 and perform sample collection
  12. The samples are left at 4 °C for 24 hours.
  13. The relative GFP intensity is taken with FACSCalibar flow cytometer.


Discussion of General Trends Found

The positive control showing a weak signal can be explained by the GFP forming inclusion bodies in high concentrations. The inclusion bodies would be fluorescently inactive and show a negative GFP signal. This theory is also support by Figure 1.C which shows two peaks for two time points. At the earlier time point, the negative GFP peak is weak while the positive gives a strong signal, however at the later time point the negative GFP signal is stronger and the positive GFP. There is also roughly the same area under both peaks meaning that it is likely that the counts from one event are being transferred to the other. Since time is the only factor and GFP accumulates over time, it can be inferred that at high concentration the cell forms the fluorescently inactive inclusion bodies. The large increase from an aTc concentration of 10 ng to 50 ng that is observed is likely explained by the saturation of TetR. With the TetR bound to its inducers, it wouldn’t bind to the operator site and produce a greater GFP intensity. However, the rate of GFP production in the TTL construct seems to be governed more strongly by the Lac operator site. This is likely due to LacR being a very large molecule which prevents polymerase from binding to the operator site. The mutations in the Tet operator site were made to change the binding affinity of TetR for the operator site. These alterations not only affect the binding affinity of just the repressor protein but also the inducer-repressor protein complex. This means that not only the leakiness of the promoter will be affected. The efficiency of repression is also affected. Our results from the AND gate expression did not support what was found when just studying just mutations in the Tet operator . The highest expression was observed with mutant 4 while mutant 3 and 5 had a slight decrease in efficiency of transcription.


TTL T0P10
IGem 2.jpg





IGem 1.jpg




Safety

1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?
No, our constructs are simple promoter regulatory elements, built from well characterized lactose and tetracycline operon components, and characterized in non-pathogenic strain of E. coli.
2. Is there a local biosafety group, committee, or review board at your institution?
Yes, there is an Institutional Biosafety Committee at the University of Minnesota. We have described our work and gotten permission to work in a wet lab (IBC Code Number: 0706H11321).
3. What does your local biosafety group think about your project?
We use standard molecular biology techniques. IBC readily approved a continuing review.
4. Do any of the new BioBrick parts that you made this year raise any safety issues? If yes, did you document these issues in the Registry?
No, none of the parts we built raise any safety issues.


Notebook

This calendar contains a day-by-day catalog of what we did in the wet lab for our project and parts characterization. The calendar for computational work can be found below and on the Modeling page. Please click on each event to see a detailed description of what we did.



June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31