Cloning

In order to design our circuits for testing the enzymes and thermometers outlined on the Project Design page,we carried out the following process:

1. Obtained the linear DNA templates for the parts and added BsaI and Esp3I overhangs
2. Inserted the part into a vector using Golden Gate Assembly to make a part plasmid
3. Performed multi-part Golden Gate Assembly to make gene cassettes of the circuits

RNA Thermometer Characterization

Testing rationall designed thermometers in time course

Motivation and Objective

In order to get familiar with the protocol of testing thermometers, we tested the thermometers that were made using a ‘rationally designed’ process. The rationally designed thermometers were created by manually adding a removing base pairs in the complementary sequence to the RBS to lower the melting points.

Method

We inoculated 2mL of pIG015, 016, 018, 019, 029 in LB30 enriched with 1mM of MgSO4. These were left to grow for 8 hours at 37°C in a 96 well block. We measured the OD600 of the saturated culture in a 1:30 dilution and then diluted the saturated culture to a final OD600 of 0.01. These were then grown for approximately 2 hours until their OD600 hit 0.1. In the meantime, Cornin 3904 black clear flat bottom 96 well plate were prepped by added 100µL of 2 mM IPTG LB. 100µL of each culture was added to the appropriate well. The plate was then grown in the plate reader at 30°C for 6 hours. This entire process was repeated the next day, but the plate was grown at 25°C for 6 hours instead.

Results and Conclusion

While we gained a good deal of experience doing this experiment, the results were rather useless. There was significant cross contamination between wells since too shallow 96 well blocks were used. The only general trend we got out of it, however, was the fact that almost none of these thermometers showed any significant improvement to the controls. We decided to abandon these ‘rationally designed’ thermometers.

Testing in solid culture using spot inoculation

Motivation and Objective

We decided to do a solid culture test of the first round of thermometer in order to get a visual representation of the fluorescence changes across temperatures.

Method

1 µL of saturated culture from each thermometer was spotted onto a LB Kan plate. Images were taken using a macroscope. The thermometers that eventually showed to be promising have been renamed. All others retain the old naming conventions.

Results and Conclusion

The images turned out to be rather difficult to quantify, but general observations were able to be made.

Measuring only the end time point: Round 1

Motivation and Objective

Method

Results and Conclusion

Thermometer time course over promising thermometers

Motivation and Objective

Method

Results and Conclusion

Measuring using ramping temperatures

Motivation and Objective

Method

Results and Conclusion

Measuring only the end time course: Round 2

Motivation and Objective

Method

Results and Conclusion