Team:Newcastle/Metal Sensor planB

=Metal Sensor: czrA+arsR=

Purpose and Justification
If our project is to process cadmium and not other metals, we need to genetically engineer Bacillus subtilis to carry out a set of cellular processes based on the action of metal sensors. These metal sensors will detect cadmium through a system known as AND Gating. There are two metal sensing repressors, which are known to respond to cadmium: arsR and czrA. By placing binding sites to these two metal sensing repressors next to each other in a promoter region, the gene regulated by that promoter will be synthesized only when a combination of metals that bind to both sensors are present; this is a combinatorial approach for gene expression regulation. Plan B allow us to meet a Gold metal criteria: we would improve BBa_J33206 (ArsR) BioBrick.

Construction

 * 1- Amplify by PCR czrA gene with its promoter and RBS region from Bacillus subtilis 168 genomic DNA. The primers will have standard BioBrick restriction sites in its dangling ends. Forward primer (primer1) with EcoI and Xbal restriction site and reverse primer (primer2) with SpeI. - the region to be amplified: 500bp upstream the start codon + czrA CDS 321bp + terminator sequence.






 * 2- Cut BioBrick part BBa_J33206 (ArsR) with EcoRI and Xbal and the amplified czrA gene with EcoRI and SpeI
 * 3- Ligation - BBa_J33206 (ArsR) + czrA gene
 * 4- Amplify by PCR the product with primer 3 and 4. Primer 3 is the forward primer, anneals at the start of arsR CDS and has got NheI restriction site at dangling end. Primer 4 is the reverse primer, anneals to the end of arsR binding site and has got BamHI restriction site.
 * 5- Amplify by PCR cadA promoter and RBS region from Bacillus subtilis 168 genomic DNA. Forward primer will have BamHI restriction site at its dangling end, reverse primer will have NheI.
 * 6- Ligation – products of step 5 and 6 are treated with NheI and BamHI and ligated.