Part:BBa_K2005060

GFP (UV-resistant)

Green red fluorescent protein (GFP) with its coding DNA sequence redesigned to minimize its susceptibility to UV-induced DNA mutation. Compared with standard GFP, this gene is therefore more stable and less likely to evolve out of a population of cells.

Sequence Design

To generate this artificial DNA sequence, we employed our custom-made algorithm for modifying gene sequences to eliminate mutagenic sites. These sites were identified based on prior research on DNA irradiation, which identified dipyrimidine motifs as particularly vulnerable. At these motifs, synonymous codon substitutions were made to eliminate pyrimidine tracks and replace them with ones with a lower predicted mutation risk. In addition, our algorithm used heuristics to factor in the effect different codons have on gene expression to ensure that the gene not only produced the same protein but produced it at similar levels. For this sequence, our algorithm could eliminate 57% of predicted irradiation sites.

Assembly

This sequence was synthesized and ligated into pSB1C3. We confirmed that this construct was synthesized and integrated correctly by sequencing. We cloned this gene with a T7 promoter (K2005061) to verify that both the function and expression of the fluorescent protein. It expressed at comparable levels to control E0040 GFP, indicating no major functional-disruption from sequence optimization.

Mutagenicity

We quantified levels of UV-induced DNA damage. Comparison to the control Bba_E0040 GFP verified that our optimization of this sequence reduced the rate at which mutagenic DNA lesions formed post-irradiation. With antibodies raised against irradiated DNA, we measured levels of pyrimidine dimers on blots of our optimized GFP constructs. K2005060 showed fewer dimers form upon equal DNA irradiation, and although our current sample size is insufficient to determine significance, we showed a significant effect with a similar construct (K1673101).

References

Senthilkumar, K., Grozema, F.C., Guerra, C.F., Bickelhaupt, F.M., and Siebbeles, L.D.A. (2003). Mapping the sites for selective oxidation of guanines in DNA. J. Am. Chem. Soc. 125, 13658–13659.