1. You have discovered a novel sigma factor in coli cells called ?25 Based on da
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1. You have discovered a novel sigma factor in coli cells called ?25 Based on data from a DNase foot-printing assay you have determined that ?25 binds to consensus sequences at positions -15 and-30 upstream of genes involved in the stress response to lambda bacteriophage infections, including gene z. You hypothesize that in order for gene z to be transcribed the ?25 protein must bind to the -15 and -30 regions. What approach could you use to test this hypothesis? You have already cloned the promoter region (containing the -15 and -30 regions) and the coding region for gene z into pUC19. What molecular techniques could you use to test your hypothesis? What results would you need to get to support your hypothesis? (4 points)Explanation / Answer
Transform an Ecoli stain which is gene z minus and ?25 plus, with pUC19 plasmid containing gene Z and the -15 to -30 region.
Transform another Ecoli stain which is gene z minus and ?25 minus with the same plasmid.
Infect both the transformed strains with lysogenic lambda bacterophage.
After infection do blue white screening with the two strains.
In the an Ecoli stain which is gene z minus and ?25 plus, the ?25 protein can bind to -15 to -30 in the plasmid and can start the expression of lac Z component which can produce functional beta galactosidase enzyme. So screening with X gal and IPTG will give blue colonies
In the an Ecoli stain which is gene z minus and ?25 minus there is no ?25 protein to bind to -15 to -30 in the plasmid, so no lac Z component is produced in this strain, hence it will not contain functional beta galactosidase enzyme. So screening with X gal and IPTG will give white colonies
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