Team:Cambridge/Experiments/Initial Exercise Group A

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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 good fun.

Ftsz was first identified in a mutant screen in 1980 (Lutkenhaus, Wolf-Watz and Donachie) as a gene recquired for bacterial cytokinesis (cell division). It is known that a ring structure partly composed of this protein forms around the cell equator at division. It was hoped that a glowing green ring would therefore be visualisable through confocal microscopy as a result of our fusion.

Notes

Template:Reflist

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.

Template B. subtilus genome
FWD Primer ccaattaaaggaggaattcaaaATGTTGGAGTTCGAAACAAACAT Order Code: 050
REV Primer agtgaacagctcttcgcctttacgGCCGCGTTTATTACGGTTTC Order Code: 060


2. GFP side

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

Template Vector Plasmid.
FWD Primer AGAAACCGTAATAAACGCGGCcgtaaaggcgaagagctgttcact Order Code: 070
REV Primer provided Code: B


3. Promoter side

Amplify fragment containing promoter (LHS on above)

Template Vector Plasmid.
FWD Primer provided Code: A
REV Primer ATGTTTGTTTCGAACTCCAACATtttgaattcctcctttaattgg Order Code: 080

Restriction Mapping

The nucleotide sequence of our insert and the plasmid used were obtained from the literature. These were collated in the plasmid-editing program 'aPe' and all restriction sites for which enzymes were available mapped.

Team1.jpg

A particular enzyme was chosen from the map that conveniently cut in three places to give fragments of a good size to be resolved on a gel. The expected gel results were modelled using the same program.

Cam team1a.jpg