Linear DNA synthesis

Linear DNA synthesis of the plant growth promoting enzymes

We obtained IaaMH by taking the DNA from the BBa_K515100 biobrick found in the kit. In order to add the correct overhangs, we did 3 separate PCRs using primers oIG005/ oIG006, oIG007/oIG008, oIG009/oIG010.. Acds was obtained by ordering a gene block on IDT. OtsBA was obtained by amplifying DNA off of the E. coli genome using oIG042/oIG043. Sequences for these primers and parts can be found on the parts page.

Linear DNA synthesis of the inducer

After doing preliminary research, we decided that the best inducer to use was araC and arabinose, since P. putida can not catabolize arabinose. We also decided to use the origin RK2 since it was well documented to perform well in P. putida. We acquired a plasmid called pBEST which contained Pcon-araC. We designed primers to amplify Pcon-araC from this plasmid and purified the PCR product. After several gel purification failures, we decided to look at the linear DNA sequence and its primers, and we discovered that the primers bind nonspecifically to other terminators in the template. We continued to troubleshoot for several days with no luck, so we ordered a gBlock of Pcon-araC.

DNA Assembly

Through Golden Gate assembly, we attempted to ligate araC into the vector pSPB440 with AmpR, RK2 origin, and a sfgfp dropout. After trying to get colonies from a vector containing araC and RK2 and failing for two months we ran an experiment with positive controls. We built a series of 4 vectors containing a sfgfp dropout and amp resistance and either araC in the vector or a double terminator, which served as the control, and either RK2 or p15A as the origin. The results indicated that only those with the p15A origin produced the correct colonies. This told us that the problem was not with ouraraC but instead was with the RK2 origin we were using. Therefore, we switched to another broad host origin.

RNA Thermometer Assembly

The thermometers were assembled by performing a PCR off of a plasmid that contains the RBS BBa_B0034. The primers used in the PCR contained the necessary overhangs needed to assemble the PCR product with the thermometer synthons. The thermometer synthons were constructed by phosphorylated oligos and then performing a four part oligo annealing reaction. We ordered 2 unique oligos for each different thermometer and were able to use two common oligos as well. The PCR product was assumed to be correct after gel electrophoresis. That same plasmid that served as the reactant for the PCR was then used as our control during the thermometer experiments.

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.

Motivation and Objective

Method

Results and Conclusion

Motivation and Objective

Method

Results and Conclusion

Motivation and Objective

Method

Results and Conclusion

Motivation and Objective

Method

Results and Conclusion

Motivation and Objective

Method

Results and Conclusion