Difference between revisions of "Part:BBa K620000"

Revision as of 03:48, 29 September 2011

DDT Dehydrochlorinase

Also known as Glutathione S-transferase 1-1, this protein is supposed to degrade DDT, an endocrine disruptor and persistent organic pollutant. The protein was identified from the Anopheles dirus mosquito by [http://www.sciencedirect.com/science/article/pii/0965174895000909 Prapanthadara et al] and the gene was assembled from the protein's amino acid sequence, taken from [http://www.ncbi.nlm.nih.gov/protein/Q93113.1 NCBI's protein database], via PIPE cloning.

Usage and Biology

Plasmid map of K620000 in pSB1C3 (submission plasmid)

Plasmid map of K620000 in pET11-a to amplify and purify the protein.

DDT in reaction buffer, original peak at 235g/mol

DDT Molecular Structure

DDT structure after GCMS with a molecular weight of 235 g/mole

12.5 minute peak of DDT reaction with DDT Dehydrochlorinase analyzed with GCMS. There is a peak at 105 g/mol and a peak at 133 g/mol.

16.5 minute peak of DDT reaction with DDT Dehydrochlorinase analyzed with GCMS. There is a peak at 191g/mol and a peak at 206 g/mol

DDT structure after reaction with DDT Dehydrochlorinase and GCMS with a molecular weight of 206g/mole

As shown in the GCMS of DDT without the degradation enzyme, there is a clear band at 235 g/mol. DDT's molar mass is 355. This indicates that the original structure loses a carbon and three chlorines to form a new structure with this mass during the GCMS process. The 12.5 minute GC peak and the 16.5 minute GC peak in the DDT-DDT dehydrochlorinase reaction show a further degradation of DDT. The 16.5 minute peak showed an MS peak at 206 g/mole. The degradation that could result in this weight involves the loss of two chlorines and a phenyl group. There is another mass spec band in this reading at 191 g/mol, which we are still working to identify. In the 12.5 minute GC peak, there are MS peaks at 133 g/mol and at 105 g/mol, which we are also still working to identify. However, these two GC peaks are not present in analysis of our reaction buffer with DDT and our reaction buffer with enzyme, indicating that they result from a degradation reaction between DDT dehydrochlorinase and DDT.