Experiment 1: Extraction of Bacilius subtilis genomic DNA
Adam Denyer, Tanel Ozdemir June 13, 2014
Protocols DNA extraction
Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including B. subtilis and P. aeruginosa. We were able to obtain a B. subtilis strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the B. subtilis genomic DNA.
Experiment 2: Transforming E. coli with Azo-reductase plasmids
We were gratefully provided with a set of plasmids from a group of researchers working at the University of Lisbon, Portugal who are researching how azo-reductase enzymes function and were keen to collaborate with us. These plasmids contained a number of azo-reductase genes from both B. subtilis and P. aeruginosa including mutated forms found to exhibit enhanced reductase activity. As the DNA concentration of the plasmids we were sent was insufficient to perform PCR amplification on we transformed each of these plasmids into our E. coli NEB5lpha competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.
Experiment 3: Diagnostic digest of azo-reductase plasmids
We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expresing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.