Difference between revisions of "Team:WashU StLouis"

For the last century as a consequence of industrialization, greenhouse gas emissions have depleted the ozone layer, especially at the Earth's poles. One function of the ozone layer is to absorb UV radiation emitted by the sun, protecting life on Earth from its harmful effects on DNA. When exposed to UV, DNA tends to form pyrimidine dimers which interfere with DNA replication and translation and can lead to mutations and cell death. While many focus their attention on the effects of UV radiation on humans, photosynthetic organisms must also be considered because they are responsible for the world's oxygen and form the basis for nearly all food chains. Though many photosynthetic organisms already have UV repair mechanisms, it is becoming increasingly necessary to fortify and supplement these mechanisms because of the drastic increase of UV exposure within the last century.

In order to protect against increased levels of UV-B radiation, our team turned to an extremely radiotolerant species of tardigrade, R. varioenatus. Known for its ability to survive in even the most extreme of environments, this strangely adorable microorganism has several genes that encode resistance to the dangers of UV irradiation. We see this as having two major applications: (1) the production of UV-resistant cyanobacteria which could replace current wild-type cyanobacteria in order to halt the cycle of ozone depletion; and (2) improving the efficiency of cyanobacteria-focused biofuel production. We then created BioBrick-compatible genetic constructs from two of these genes and expressed them in E. coli and cyanobacteria as a proof-of-concept.

Our project has several components. The first of these is simply to compare the effectiveness of several genes on the level of UV radiation tolerance in E. coli. The first gene is uvsE, an endonuclease triggered by UV damage, which originates from Deinococcus radiodurans, an extremophile that is known to be one of the most radiation-resistant organisms in existence. This gene has already been characterized by another iGEM team and is easily accessible in a plasmid. Two of the genes are derived from Ramazzottius varieornatus, a species of tardigrade, which are organisms known for their extraordinary resistance to extreme conditions. One of these genes is Dsup, a DNA-binding protein which was recently discovered and has been shown to protect against ionizing radiation; however, no studies have yet been published on its effectiveness in protecting against UV radiation. The other tardigrade gene that will be tested is phrA, a photolyase. The tardigrade photolyase is a homologue of our final gene, the photolyase that exists in the cyanobacteria genus Synechococcus. In addition, we will be experimenting with a UV-induced promoter and plasmids with different copy numbers to see if these constructs are more efficient. After transforming these genes into E. coli, we will be transforming cyanobacteria with our gene constructs with the hope of seeing the intended effect in cyanobacteria. In order to test the efficacy of these genes, we will expose the transformed E. coli and cyanobacteria to UV light through a UV exposure apparatus we developed.

Throughout our work over the summer we accomplished a number of important tasks towards realizing the goals we set forth in our plan. First, we were able to confirm that the Dsup gene taken from R. varioenatus does in fact protect against UV radiation. Through the testing of our genetic constructs with the Dsup gene, we produced data to support that Dsup protects against UV-B radiation and also illustrated the significant change in UV-B resistance that can be given to E. Coli cells and potentially cyanobacteria. We also created a new piece of hardware - the Environmental Simulation System - which functions as a tabletop incubation chamber capable of exposing cells to UV-B radiation in both liquid cultures and plated cells.