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<h1 style = "background-color:#f7c921!important;"> Cloning </h1> | <h1 style = "background-color:#f7c921!important;"> Cloning </h1> |
Revision as of 20:50, 20 October 2019
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:
- Obtained the linear DNA templates for the parts and added BsaI and Esp3I overhangs
- Inserted the part into a vector using Golden Gate Assembly to make a part plasmid
- Performed multi-part Golden Gate Assembly to make gene cassettes of the circuits
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
Motivation and Objective
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.
Methods
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.
Resulting and Conclusion
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
Motivation and Objective
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.
Methods
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.
Results and Conclusion
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 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
P. putida Experiments
Testing P. putida F1 MIC for Carbenicillin and Ampicilin
Motivation and Objective
It was important for us to use as little antibiotic as possible, since Carbenicillin, a β inhibitor, has relatively mild effects of plant growth still affects plant growth. We set out to test how much antibiotic we could use while still maintaining selectivity of pseudomonas .
Methods
We grew both Pseudomonas transformed with a plasmid containing AmpR and wildtype in various concentrations of Carbeniciliin and Ampicillin ranging from 0.5 mg/L to 50 mg/L.
Results and Conclusions
Wild type P. putida grew in concentration of 2.5 mg/L and under of Ampicillin and Carbenicilin. We decided to use 5 mg/L of Ampicillin for cloning. This concentration is too high for plant experiments. Thus we did not use antibiotics in the plant experiments, but instead worked in a biosafety cabinet under sterile conditions.
A. thaliana Plant Experiments
With untransformed P. putida and 0.6% PN glucose
+0.01% glu
/*insert image here but let sam do this part*/+0.1% glu
With and without arabinose
10 μM
50 μM
With various concentrations of auxin
0 µM, 100nM, 1 µM, and 10 µM
With various concentrations of trehalose
0 mM, 0.5 mM, 1mM, 5mM, 10 mM
With arabinose, auxin, and trehalose at maximum concentration
Arabinose 50 mM, Auxin 10 uM, Trehalose 10 mM
With and without P. putida
in progress