Team:Rice/Experiments


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

    4. Cloning Cycle

    The cloning clycle outlines the basic procedure we used to make our parts. All protocols can be found on our Protocols page.

    1. Linear DNA synthesis
    2. DNA Assembly
    3. Transformation
    4. Solid culture (plating)
    5. Colony picking
    6. Liquid culture
    7. DNA Miniprep + Spectrophotometry
    8. Restriction digest
    9. Agarose gel electrophoresis
    10. Sanger sequencing

      11. Linear DNA synthesis

      Linear DNA synthesis of the plant growth promoting enzymes

      Initially, we obtained the desired DNA sequences that coded for our enzymes by looking at the Standard Registry for Biological Parts. We input them into Benchling, and made forward and reverse primers so that we could amplify the DNA through PCR or oligo annealing.

      Linear DNA synthesis of the promoter

      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 Pcon-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 our araC 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. 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

      P. putida Experiments

      P. putida Fluorescence

      P. putida and Carbencillin

      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