Team:Cambridge/Experiments/Initial Exercise Group Alpha

Initial Exercise: Joe, Matt, Felix and Gerrit

Fusion of SrfA promoter with GFP

Aim

Our week 1 experimental challenge was to generate a temporal and or spatial pattern of GFP expression in Bacillus subtilis, using Gibson Assembly to join a sequence of interest to a GFP coding plasmid. We chose to visualise sporulation via a fusion of SrfA promoter with GFP.

Role of SrfA

• SrfA codes for surfactin production, a protein that is produced by B. subtilis prior to sporulation.
• SrfA is regulated by ComA, a protein which itself responds to an intercellular population-density-signalling molecule, CSF, in a concentration dependent manner.
• ComA has low activity at low CSF concentration(low population density), highly active as CSF levels increase, and less active at high CSF concentrations.

Sporulation occurs late in the life cycle of B. subtilis when the colony reaches a high population density and we hope that GFP could be visualised following overnight growth.

This paper[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1178101/?page=2] details some sporulation inducing culture conditions.

Construct Design

We wish to:

• keep the original promotor for SrfA and comA boxes*
• remove GFP promotor sequence*
• maintain ComGA stabiliser sequence*
• do not include restriction sites*

Predicted results

Bacillus colonies also exhibit a range of colony patterns so we hope that superimposed on the bacillus colony shape will be
temporal pattern of green fluorescence indicating which bacteria are on their way to sporulation.

Zero or low GFP detection should be the initial condition, changing to higher GFP activity when SrfA is activated prior to sporulation. This may also mark spatially the regions of the colony where sporulation is about to occur.

Method

We designed 20bp primers with 20bp tails with which to perform gibson assembly to fuse the SrfA promoter with a plasmid containing GFP.

The primers arrived as dry pellets with datasheets that detailed the quantity of water required to make these dry pellets into stock solutions of 100uM concentration. We then made a four fold dilution to make 25uM 'working stock' solutions. We were advised to take extra care to make the inital stock amounts perfect as these affect all future dilutions and that making a working stock is useful as it means you have the capacity to repeat your experiment if anything goes awry.

We were provided with genomic DNA from B. subtilis and the vector plasmid containing GFP in two fragments. In addition we were given two primers which would combine with our designed primers to seal the two fragments.

Our three PCR tubes contained: 1. 1ul primer JMF2 1ul primer JMF3 1ul B. subtilis genomic DNA

2. 1ul primer JMF1 1ul primer A Forward (provided) 1ul vector DNA

3. 1ul primer JMF4 1ul primer B reverse (provided) 1ul vector DNA

to each tube we also added: 9.5ul of water 12.5ul Master mix (SyBR Green and Rox, Hotstart Taq polymerase, dNTPs and dyes)

to complete a total volume 25ul total

The complete tubes were run in a real time PCR machine for 30 cycles with a 2 minute extension time and a primer annealing temperature of [INSERT TEMPERATURE HERE]

Technical Data We used Finnzymes melting temperature calculator to work out the melting temperature of the primer part (not the tail) of our Gibson Assembly oligos (this was the 3' 20bp of each oligo) these values in degrees C are shown below

JMF1 - 56.91 JMF2 - 68.8 JMF3 - 50.48 JMF4 - 53.09

Results [to be written up]

References