Team:Osaka/Project

Project Details

DNA damage tolerance

D. radiodurans

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It is an aerobically-growing bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, E. coli can withstand up to 200 Gy, and an acute exposure of just 5-10 Gy is lethal to a human being.

We explored various genes from D. radiodurans, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into E. coli, we hoped to confer additional DNA damage tolerance to the host cells.

DNA damage detection

SOS response

If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly. This reaction to DNA damage is the SOS response.

RecA is a 38 kilodalton Escherichia coli protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted.

We decided to employ the promoter of the RecA gene (BBa J22106) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.