Team:University of Sheffield /Wet Lab



=Wet Lab=

Click here, or on the navigation bar, for the overall timetable of our wet lab sessions.

Click here, or on the sub-navigation bar, for almost all the protocols we used in our wet lab sessions.

The lab books below contain the more detailed jobs carried out by each member of the team.

Overview


The Diagram is a stepwise representation of a plan we wanted to carry out to achieve our goal. First of all gene coding for BarA protein should be knocked out from wild strain of an engineered bacterium (E.coli MG1655). Then GFP should be introduced into ΔbarA mutant (MGbara1) under a promoter of a gene positively regulated by BarA. Simultaneously to those steps Fusion Kinase should be created and inserted into an expression plasmid. Then both intermediates, ΔbarA mutant with GFP in its genome (MGbara2) and plasmid with Fusion Kinase, should be combined together to give rise to the biosensor (MGfusrec). Functionality of a biosensor should be analyzed by exposing it to CAI-1 autoinducers. If the design is successful the bacterium should grow.

Plasmids and Primers
Plasmids:


 * pCP20 - "an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis" (Datsenko and Wanner,[4])
 * pCQSA - plasmid sent to us by Bonnie Bassler's lab, contains the CAI-1 producing gene
 * pCQSS - plasmid synthesised by us (via idtDNA) containing the gene for the CAI-1 receptor