Team:Minnesota/Project

From 2009.igem.org

(Difference between revisions)
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<br />
<br />
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<h1>The Experiments</h1>
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=== The Experiments ===
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<h1>Results</h1>
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<h1>Protocols</h1>
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=== Part 3 ===
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These are the standard techniques that we used in the wet lab:
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<br />
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== Results ==
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<br />
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<h3>Bacterial Culture</h3>
 +
<h4>Sterile Technique</h4>
 +
<ol>
 +
<li>Always work around a flame or in the hood</li>
 +
<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>
 +
</ol>
 +
<br />
 +
<h4>Bacterial Culture Maintenance</h4>
 +
Culture cells:
 +
<ol>
 +
<li>At 36 degrees Celsius</li>
 +
<li>Shaking at 220 rpm</li>
 +
<li>At 10% total flask/tube volume</li>
 +
<li>In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x10<sup>8</sup>cell/ml)
 +
</ol>
 +
<br />
 +
<h4>Bacterial Culture For Gene Expression Experiments</h4>
 +
<ol>
 +
<li>Pick and individual colony from a plate and inoculate 2ml LB + amp media</li>
 +
<li>Incubate overnight at 37 C, shaking at 220 rpm</li>
 +
<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>
 +
<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>
 +
<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>

Revision as of 20:26, 15 July 2009

Mnlogo.jpg
Home The Team The Project Parts Submitted to the Registry Modeling Parts Characterization Notebook

Contents

The Project

ANDgate.jpg

We studied the potential of a synthetic, single promoter AND gate. This helps us understand at a fundamental level what is happening in a network with multiple regulators and opens further research paths. The device consists of parts of the Tet (tetracycline) and Lac (lactose) and responds to commonly used inducers IPTG (Isopropyl β-D-1-thiogalactopyranoside) and aTc. Three or fewer of these operators can be combined to form a promoter and the order and quantity of each operator site result in different constructs, that is, given three blank spots for operators on a promoter and the choice of either -, T or L for each spot, there are a variety of possible promoter designs. We chose to focus on four constructs:

  • T T L
  • T T -
  • T - -

We also examined 4 mutations in the Tet operator site for each construct.




The Experiments



Results


Protocols

These are the standard techniques that we used in the wet lab:

Bacterial Culture

Sterile Technique

  1. Always work around a flame or in the hood
  2. Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping
  3. Sterilize metal instruments between uses by dipping in 100% ethanol and flaming


Bacterial Culture Maintenance

Culture cells:

  1. At 36 degrees Celsius
  2. Shaking at 220 rpm
  3. At 10% total flask/tube volume
  4. In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x108cell/ml)


Bacterial Culture For Gene Expression Experiments

  1. Pick and individual colony from a plate and inoculate 2ml LB + amp media
  2. Incubate overnight at 37 C, shaking at 220 rpm
  3. Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture
  4. Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)
  5. Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture
  6. Continue cultures as described above in "bacterial culture maintenance" for 9 hrs
  7. Isolate cell samples from cultures at 3, 6, and 9 hour time points
  8. Remove 100ul sample aliquots from cultures
  9. Pellet samples at 5K rpm for 5 minutes
  10. Remove supernatant
  11. Wash cells with 1 ml chilled 1xPBS, pH 7.6
  12. Resuspend cells by vortexing
  13. Re-pellet cells at 5K rpm for 5 minutes
  14. Remove supernatant
  15. Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)
  16. Incubate at RT for 30 minutes
  17. Pellet cells at 5K rpm for 5 minutes
  18. Remove supernatant
  19. Resuspend cells in 1 ml 1xPBS
  20. Store samples at 4 C until analysis by flow cytometry
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