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From 2012.igem.org

Week 1 (May 1-4)

Week 2 (May 7-11)

Decarboxylation

Things we did.

Denitrification

In the first two weeks of iGEM our group has focused on reviewing literature regarding the bioremediation of nitrogen groups attached to naphthenic acids. The most prevalent N heterocycle is carbazole, representing 75% of total nitrogen by mass. The upper pathway of carbazole biodegradation is catalyzed by the enzymes coded for by the car operon, CarA (CarAaAbAd), CarB (CarBaBb), and CarC. These enzymes convert carbazole to anthralinic acid. The lower pathway is catalyzed by the enzymes of the ant operon, antA, B, and C, yielding cathecol while releasing CO2 and NH3. The car and ant operons are both regulated by the Pant regulator which is induced by the protein, antR. CarAa also has its own promoter which is not induced by antR. We have also investigated an alternative pathway using CarA combined with an amidase (amdA) that selectively cleaves NH2 from an intermediate of the car pathway. This could bypass much of the car/ant pathway and is possibly more efficient.

We have decided to use Pseudomonas resinovorans and Rhodococcus erythropolis to amplify these genes from. CarABC and AntABC from P. resinovorans has been shown to have a wide range of nitrogen containing substrate specificity. R. erythropolis contains the amdA gene that we wish to use, and some evidence suggests that it may also be able to degrade sulfur rings through its CarABC pathway.

In addition to our research we have also been learning some of the lab techniques we will be using this summer. This includes transforming a plasmid into E. coli, plating and selecting for bacteria containing the plasmid, verifying with colony PCR, performing a mini-prep and a restriction digest.

Desulphorization

Things we did.

Ring Cleavage

Things we did.