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.

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. As always, the thermometer cassettes were confirmed via sequencing using _____. That same plasmid that served as the reactant for the PCR was then used as our control during the thermometer experiments.

RNA Thermometer Characterization

RNA Thermometer Fluorescence Time Course Measurements