Team:Exeter/Experiments

Protocols

Competent cell preparation

Throughout the project, the E. coli cells in use needs to be genetically modified, specifically by the insertion of artificial plasmids. This transformation procedure required cells that were capable of taking up plasmids and these were prepared thus.

Transformation of competent cells

Insertion of plasmids into the competent cells we made was done in this way.

Modular cloning strategy

The individual subparts (and in the case of the operon, CDS segments) had to be brought together to create the composite parts. These individual components were all brought together in one reaction in one container. This was done using the MoClo protocol.

SDS-PAGE

SDS-PAGE and Western Blot analysis was used to characterise protein expression. For SDS-PAGE we used a ThermoFisher NuPAGE Bolt system, the principles of which are described on the following link. The ladder was SeeBlue Plus2 Pre-stained protein standard (ThermoFisher) and gels were stained with Simply Blue Safe Stain (Coomassie blue, ThermoFisher). For Western-Blot analysis we used a PiercePower Blotter (ThermoFisher) for semi-dry blotting onto PVDF-membranes. These were probed using either an Anti-His or Anti-GFP primary antibody raised in Mouse and anti-mouse alkaline phosphatase-conjugated secondary antibody raised in goat with a ThermoFisher iBind system. Membranes were then visualised using SigmaFast BCIP/NBT (Sigma).

Biofilm formation on tori

We developed over the summer a protocol for formation of biofilms on the polymer surfaces of polypropylene scaffold structures. This was done using the information given by Mike Allen at the Plymouth Marine Laboratory. The scaffold structures were used in the Metal Binding Reactor.

Testing the MBR

A process was developed for testing the efficacy of biofilm bacteria to bind to substrates in the flow of water through the metal binding reactor.

Testing the Hydrocyclone

Protocol for testing separation efficiency of the hydrocyclone can be found here.

HPLC protocol

High performance liquid chromatography was used to analyse the products of the MBR tests. The MBR run-through had to be prepared however, before entering the machine.

Biosecurity

Despite our first hand observation of the use of ultra-violet radiation as a means of sterilisation in water treament facilities, we wanted to test the effectiveness of this method. We developed a protocol which we used and also sent to the iGEM teams from Cardiff and Newcastle as part of our collaborations with them.

PCR

PCR was used regularly when we needed to amplify DNA, either for diagnostic or functional reasons.

Induction protocol

A number of our constructs contained either the P_Rha or P_Ara promoter or both. These constructs were used in a number of different strains of E. coli over the course of the project. Through experimentation with the conditions of the induction procedure, we determined the protocol most conducive to pili expression. The T7 construct was expressed using an auto-induction media outlined here.

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