Team:Cambridge/Experiments/Initial Exercise Group One

Initial Exercise: Katy, Heather, Marta and Veronica

The aim of the project was to produce interesting patterns of fluorescence in a colony of Bacillus Subtilis bacteria by fusion of a single gene of interest in the bacillus ge=nome to GFP using Gibson Assembly. These patterns would need to be clearly observable using a confocal microscope. After some research, our group decided to fuse GFP to the DivIVA gene.

Reasons for choice

We decided to perform this fusion for a number of reasons. Firstly, DivIVA plays and interesting role in cell division; it is believed to control septum positioning in Bacillus Subtilis. This was investigated (in [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1997.3811764.x/abstract this] study by Edwards and Errington) by considering the phenotype of mutants produced when certain bases in the gene were altered.

Mutants showed:

• inhibition of cell division initiation*
• abnormally placed septum*
• some small anucleate cells*

We also hoped that this fusion would have a high chance of success, since a successful DivIVA-GFP fusion had already been documented, indicating that fluorescence was constitutively expressed at all sites of cell division, new and old, in the bacteria. We therefore know what characteristics to expect and so we should be able to determine whether or not the experiment was successful almost immediately under the microscope.

Construct Design

We wish to:

• keep the original promotor for DivIVA*
• remove the GFP promotor sequence*
• remove the DivivA stop codon so that GFP is translated together with the DivIVA protein*
• Remove the ComGA stabiliser sequence pre-existing on the plasmid vector*

‘Linker’ code was also used by Edwards and Errington - however, we suspect that this is related to the restriction enzyme assembly process they used and hence we didn’t include it in our design.

Primer Design

used the Gibthon Construct Designer developed by the 2010 iGEM Cambridge team

Left side of the Div IVA coding sequence(site 1)

Right side of the Div IVA coding sequence(site 2)