Team:Rice/Design

Cloning method

Our type IIS assembly method

Circuit Design

Construct for testing the enzymes

acdS Cassette

acdS encodes for 1-aminocyclopropane-1-carboxylate (ACC) deaminase synthase. ACC deaminase helps reduce the overall concentrations of ethylene in the plant by converting its precursor, ACC, into ethylene and ammonium.

iaaMH Cassette

Both iaaM and iaaH function to synthesize indole-3-acetic acid (IAA) from tryptophan. iaaM encodes for tryptophan mono oxygenase and iaaH encodes for indoleacetamide hydrolase.

otsBA Cassette

Both otsB and otsA function to synthesize trehalose synthase from glucose molecules. otsA encodes for trehalose-6-phosphate synthase and otsB encodes for trehalose-6-phosphate phosphatase.

Construct for testing the thermometers

We had originally sought to use a vector that had RK2 with araC for both the thermometer and enzyme testing. RK2 has been reported to function well in P. putida, but after 2 months of trying, we performed control experiments of various different combinations of inducer and origins, and the only one that consistently would not clone was RK2 and araC. Because of this, we chose the broad host origin RSF1010. LacI was used temporarily as we were trouble shooting araC, but we decided to keep it in the system in order to be consistent.

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.