Revision as of 22:56, 19 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

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

RNA Thermometer Fluorescence Time Course Measurements

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

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 <h1 style = "background-color:#1f9ec1!important;"> <i>P. putida</i> Experiments</h1>
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-<h2><i>P. putida</i> Fluorescence</h2>
 <div class="row">
 <h2>Testing P. putida F1 MIC for Carbenicillin and Ampicilin </h2>

Difference between revisions of "Team:Rice/Experiments"