We began our project design by researching genes that had the potential to confer resistance against UV-B radiation. In the end, we decided to test four different genes from three different species.
We chose to test uvsE because it had been previously tested by another iGEM team; we intended to use this as a baseline to compare to our other genes. The homolog of phrA found in R. varieornatus was chosen because it has been shown to play an important role in UV resistance in tardigrades. The homolog of phrA found in cyanobacteria was chosen to be used as a comparison to the other genes when transformed into E. coli. Finally, Dsup was chosen because it had previously been shown to protect against ionizing radiation, but had no known mechanism of repair and had not been tested on non-ionizing radiation.
The four genes collectively utilize at least two different mechanisms of protection against UV-B radiation. uvsE is a nucleotide excision repair gene, while the phrA homologs are photolyases. Although the mechanism of repair for Dsup is currently unknown, it has been hypothesized to bind to free DNA and prevent damage from occurring in the first place. Additionally, Dsup was recently determined to protect against damage by oxidative stress.
The four genes were cloned into constructs that also included an inducible Lac promoter and a reporter, blue chromoprotein.