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"
!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/Modeling|<font color="gold">Modeling</font>]]
 
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</font>]]
!align="center"|[[Team:Minnesota/Designer|<font color="gold">SynBioSS Designer</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/Modeling|<font color="gold">Modeling</font>]]
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!align="center"|[[Team:Minnesota/Notebook|<font color="gold">Experiments and Calendar</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">Competition Requirements</font>]]
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<h2>Overview</h2>
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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.
 +
<h2>Standard Protocols We Used in the Wet Lab</h2>
<h2>Standard Protocols We Used in the Wet Lab</h2>
<h5>These link to pdf files:</h5>
<h5>These link to pdf files:</h5>
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[[Media:Bacterial_Culture.pdf|Bacteria Culture Protocols]]
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[[Media:Bacterial_Culture.pdf|Bacterial Culture Protocols]]
[[Media:Transformation_of_Chemically_Competent_Cells.pdf|Transformation of Chemically Competent Cells]]
[[Media:Transformation_of_Chemically_Competent_Cells.pdf|Transformation of Chemically Competent Cells]]
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[[Media:Sequencing.pdf|Sequencing]]
[[Media:Sequencing.pdf|Sequencing]]
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[[Media:Preparing_Competent_Cells.pdf|Preparing Competent Cells]]
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[[Media:Soeing_PCR.pdf|SOEing PCR]]
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[[Media:Ligation_Reaction.pdf|Ligation]]
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[[Media:Screening.pdf|Screening]]
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[[Media:Sample_Collection.pdf|Sample Collection]]
<br />
<br />
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<h2>Procedure</h2>
 +
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<ol>
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<li>[http://2009.igem.org/wiki/images/4/4a/Preparing_Competent_Cells.pdf Prepare Competent Cells] for TOP10 and DH5αPro</li>
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<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>Combine [http://2009.igem.org/wiki/images/a/ae/Soeing_PCR.pdf Soeing PCR] reagents with primers and place in a thermocycler.</li>
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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>
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<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>
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<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>
 +
 +
<br />
 +
<h2>Discussion of General Trends Found</h2>
 +
 +
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.
 +
 +
 +
 +
[[Image:IGem_4.jpg|350px|left|TTL T0P10]][[Image:IGem_2.jpg|350px|right]]
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[[Image:IGem_1.jpg|350px|center]]
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<h2>Safety</h2>
 +
1. Would any of your project ideas raise safety issues in terms of:
 +
researcher safety, public safety, or environmental safety?
 +
<br />
 +
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.
 +
<br />
 +
2. Is there a local biosafety group, committee, or review board at your institution?
 +
<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 />
 +
3. What does your local biosafety group think about your project?
 +
<br />
 +
We use standard molecular biology techniques. IBC readily approved a continuing review.
 +
<br />
 +
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?
 +
<br />
 +
No, none of the parts we built raise any safety issues.
 +
<br />
<br />
<html>
<html>
<body>
<body>
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<h2>Our Google Calendar</h2>
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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 on the<a href="http://2009.igem.org/Team:Minnesota/Modeling">
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<h2>Notebook</h2>
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Modeling</a> page.
+
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  
 +
<a href="http://2009.igem.org/Team:Minnesota/Modeling">Modeling</a> page.
Please click on each event to see a detailed description of what we did.  
Please click on each event to see a detailed description of what we did.  
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|The Minnesota iGEM '08 team consists  of groups working on two different projects. One team is located primarily in Minneapolis and is designing a comparator for their project. The other team is based in St. Paul and is working on a time bomb.
 
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|[[Image:Minnesota.gif|200px|right|frame]]
 
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'''Team Comparator'''
 
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|Control systems are an integral component of almost all aspects of life. Whether it is in industrial, biological, or chemical applications, controllers provide a way to keep systems functioning properly. A vital part of any control system is the comparator. This component compares a set point value and a measured value, and determines which is larger. It then sends the appropriate signal to the controller, which reacts to bring the system back to the set point. In typical applications, the controller equipment is electronic. However, our team set out to create a comparator using only genetic components. This comparator could potentially be used as part of a new, solely biological control system that could be used to treat many diseases afflicting humans, for example diabetes. This comparator could compare the blood sugar of a patient to what it should be, and send this result to a control system that could compensate, for example by changing the levels of insulin.
 
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In order to undertake this task, a system involving six genes was designed. For our system, the two inputs (one representing the set point and one representing the measured value) are IPTG and ATC. These inputs will activate the transcription of the LacI and TetR proteins, and set in motion the rest of the system to produce the outputs. Depending on the amounts of the two inducer molecules added to the system, either green fluorescent protein (GFP) or red fluorescent protein(RFP) will be produced. The actual design of the system can be seen below. In order to complete this project, a total of six genes will have to be cloned into plasmids, and two new BioBrick parts will be produced. One will be a TetR and p22-MNT dual-repressed promoter, and the other will be a LacI and lambdaphage cI dual-repressed promoter. We will also build mathematical models and conduct computer simulations that will help with the designs. This project will pave the way for other parts of a true genetic PID controller to be produced, which could be an exciting scientific development in the near future.
 
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|[[Image:Team_Pic_Minnesota.jpg|300px|right|thumb|Team Comparator]]
 
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<!--- The Mission, Experiments --->
 
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{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="90%" align="center"
 
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!align="center"|[[Team:Minnesota|<font color="gold">Minnesota Home</font>]]
 
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!align="center"|[[Team:Minnesota/HomeComparator|<font color="gold">Team Comparator Home</font>]]
 
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!align="center"|[[Team:Minnesota/TeamComparator|<font color="gold">The Team</font>]]
 
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!align="center"|[[Team:Minnesota/ProjectComparator|<font color="gold">The Project</font>]]
 
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!align="center"|[[Team:Minnesota/PartsComparator|<font color="gold">Parts Submitted to the Registry</font>]]
 
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!align="center"|[[Team:Minnesota/ModelingComparator|<font color="gold">Modeling</font>]]
 
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!align="center"|[[Team:Minnesota/NotebookComparator|<font color="gold">Notebook</font>]]
 
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==Notebook==
 
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<br><br>
 
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{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="10" bordercolor="#00CCFF" width="62%" align="center"
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!align="center"|{{#calendar: title=Minnesota |year=2008 | month=06}}
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!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=06}}
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!align="center"|{{#calendar: title=Minnesota |year=2008 | month=07}}
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!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=07}}
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!align="center"|{{#calendar: title=Minnesota |year=2008 | month=08}}
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!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=08}}
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!align="center"|{{#calendar: title=Minnesota |year=2008 | month=09}}
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!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=09}}
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!align="center"|{{#calendar: title=Minnesota |year=2008 | month=10}}
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!align="center"|{{#calendar: title=Minnesota-experimental |year=2009 | month=10}}
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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