"
Team:Calgary/Project/Promoter/Bioinformatics
From 2011.igem.org
The Project
NA-Sensitive Promoters using Bioinformatics and qRT-PCR
Project Background
A recent survey (footnote: Del Rio) found that two bacterial strains LD1 and LD2 (later identified as Pseudomonas fluorescens and putida) were capable of almost completely degrading a mixture of naphthenic acids when co-cultured together.
The above image shows the
insert the bit here about the bioninformatics search dark horse and all come one pplllllllll!!!!!!!!!!!!!!1
Using RT-qPCR to Detect Upregulation of Genes in Response to NAs
The pseudomonas strains (P. putida and P. fluorescens) were exposed to NAs to see if the genes selected through the bioinformatics search were in any way upregulated when compared with ordinary levels of expression. If they were upregulated it would indicate whether or not they contained NA responsive regions and hence a possible NA responsive promoter.
After treating the cells they were then preserved by exposure to an RNA stabilizing chemical, lysed and the RNA extracted. Next the RNA was converted to cDNA using random short primers and reverse transcriptase. The cDNA was then subjected to RT-PCR with primers that would amplify select genes of interest as well as control primers that amplify 16S-RNA sequences whose levels were expected to be stable.
To date no data has been obtained from the PCRs because the primers have not been amplifying the genes. Trouble shooting and optimization are ongoing at this point to determine the best parameters for the primers to work. MAKE CHANGES HERE NEW DATA HAS BEEN OBTAINED !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
