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: (make it a numbered list) (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

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

    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.

    DNA Assembly

    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

    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