Team:Cambridge/Experiments/Initial Exercise Group A

Initial Exercise: Cat, Jonathan, Haydn and Ai

As a 'warm-up' exercise to acquaint the group with molecular biological laboratory techniques, three mini-teams were tasked with creating an interesting GFP fusion. Group A decided that visualing ftsZ in real time, in vivo would be rather cool.\

Ftsz was first identified in a mutant screen in 1980 <ref>(Lutkenhaus, Wolf-Watz and Donachie)</ref> as a gene recquired for bacterial cytokinesis (cell division).

Notes

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Primer Design

The plasmid vector we were supplied with contains a strong promoter upstream of a sfGFP coding sequence. Our fusion design relies on amplifying the ftsZ coding region from Bacillus and creating regions of overlap between this and the GFP coding sequence in the plasmid, in order to create the gene fusion.

The desired end product is shown below, with the plasmid in lowercase and the ftsZ coding region insert in upper case.

ccaattaaaggaggaattcaaaATGTTGGAGTTCGAAACAAACAT-----AGAAACCGTAATAAACGCGGCcgtaaaggcgaagagctgttcact
ggttaatttcctccttaagtttTACAACCTCAAGCTTTGTTTGTA-----TCTTTGGCATTATTTGCGCCGgcatttccgcttctcgacaagtga

Returned Primers

The Technical datasheet we received

Procedures

PCR

Three separate PCR reaction were required, detailed below. These reactions were carried out using the automatic PCR machine.

1. ftsZ

Aim is to amplify ftsZ.

2. GFP side

Amplify fragment containing GFP coding sequence (RHS on diagram above) & part of vector plasmid

3. Promoter side

Amplify fragment containing promoter (LHS on above)

Restriction Mapping