Team:IISER-Mohali/Experiment

 

Experiments

Research has shown that cat urine contains pheromone precursors and major urinary proteins which mice brains are biologically programmed to perceive as a threat, scurrying them away at the slightest detection. Using synthetic biology, our team has reconstructed the cat felinine biosynthetic pathway in yeast(Saccharomyces cerevisiae).

This involved cloning of the parts which we obtained from iGEM Parts Registry, on plasmids (with given markers) followed by transforming these in Saccharomyces cerevisiae and finally checked the presence of Felinine via HPLC. The vectors which we would use in our setup are the parts BBa_K1362091, BBa_K081006, BBa_K1362095, BBa_K1362097 pRS313 and pRS314. These plasmids contain different origins of replication, thus resulting in different copy numbers of the corresponding plasmid in Saccharomyces cerevisiae. The copy number influences the amount of protein produced by the cell and plays a crucial role in our design. Depending on the function of a particular protein in our system, we evaluated on which plasmid the corresponding gene should be cloned.

Gene Circuit

The circuit can be divided into 3 parts. The first one is constitutive promoter – TEF, then the gene GSTM3, with HA tag on its N terminus and FLAG tag on its C terminus and the cyc terminator.

Codon was optimized for expression in S. Cerevicease because this was a cat gene.

Cloning Strategies

The gene was custom synthesised and amplified using taq polymerase

Cloning of GSTM3 in pRS313 vector using Xba1 and EcoR1

Vector Map

Vector Map with insert

Western Blot

The gene was custom synthesised and amplified using taq polymerase

Cloning of GSTM3 in pRS313 vector using Xba1 and EcoR1

HPLC

The gene was custom synthesised and amplified using taq polymerase

Cloning of GSTM3 in pRS313 vector using Xba1 and EcoR1