Team:Warwick/Experiments

Experiments

Initially we began by splicing gene fragments provided by iGEM - due to the complexity of our part, we found it to be a lengthy process and time was running out. Professor John McCarthy suggested we use Gibson assembly, providing us space in his lab and assistance from his colleagues.

In late October when the fragments were due to arrive, preparations for the assembly revealed that the backbone the fragments had been prepared for was Kanamycin; however the iGEM registry only accept submissions from Chloramphenicol resistance marked backbones (pSB1C3 specifically).

In order to fix this, primers were prepared which would match the ends of the pSB1C3 linearised backbone. Tails on these primers had the same sequence as the tips of the Kanamycin backbone. By performing PCR, the pSB1C3 backbones could be modified in such a way to allow for Gibson assembly directly into the correct backbone.

Three experiments had been considered for the modified cells:

A. Grow the cells on an IPTG gradient plate: Cells in greater concentration of IPTG should form thick biofilm, and cells in lower concentrations of IPTG will form thinner biofilms.

B. Grow the cells on an IPTG spot plate: Cells are to be spread over a plate and a drop of IPTG is to be placed and marked. Cells around the droplet of IPTG should form a thick biofilm, and cells in thinner areas will form less (E.G Part:BBa_K777121 BBa_K2012002)

C. Grow the cells on a lactose plate: A plate with Glucose, Lactose, and Arabanose is prepared. First the cells will degrade glucose - biofilm will be thin. Cells will then degrade Lactose - biofilm will become thicker. Cells will then degrade Arabanose - biofilm will become thin once more. By using a camera to perform a time-lapse, the Biofilm Manipulator's ability to control cellular adhesion and motility would be clearly defined.

(Positive and negative control plates containing a constant IPTG concentration and no IPTG respectively should be used for comparison)