Team:Exeter/Lab Introduction

Pili+

Modified pili expression

We wanted to produce modified pili to primarily bind a variety of metal ions but also with reporters to allow easy verification of protein expression. A two plasmid system was designed where the modified FimH would be expressed from one plasmid and the remaining proteins in the fim operon expressed from the second plasmid. The two plasmids carried compatible origins of replication (pUC and p15A) and different antibiotic resistance genes (AmpR and CmR) to allow for co-transformation in E. coli (Fig.1)

Fig.1 Shown are two examples of the plasmids we created. On the left is the plasmid designed to produce all the proteins of the fim operon apart from FimH. The example specifically shown has the operon under control of an arabinose inducible promoter and is in a low copy number plasmid to avoid overexpression and metabolic burden.The plasmid on the right is an example of a FimH fusion protein under control of a rhamnose inducible promoter. This is made to produce simply the modified FimH protein with an sfGFP or metal binding domain fused at the 1st amino acid position (after the signal peptide).

The FimH protein was modified to carry either sfGFP (reporter), 6xHistidine tag (reporter and metal binding) or two different metallothioneins (metal binding) (Fig.2a). See Basic Parts page for information. The Fim operon consisting of six proteins with native RBS (Fig.2b). Four different promoters were chosen to allow investigation of expression in a range of E. coli chassis. See Composite Parts page for information. Together this design gives a collection of parts for modified pili production.

Fig.2This schematic outlines the subparts of one example of our composite parts. The rhamnose inducible promoter is used in conjunction with the strong and well characterised B0034 RBS and B0015 terminator. The coding sequence is for a FimH-sfGFP fusion protein.

Hydrocyclone

Metal Binding Reactor

Biosecurity

The purpose of this experiment was to test the effectiveness of using UV as a bactericide to determine if it was suitable to be used within our filtration system as our biosecurity mechanism. The idea originated through our communications with South West Water who employ UV to remove naturally occurring bacteria that are used in their works to digest organic waste. As we are using genetically modified bacteria we felt it necessary to experimentally test its effectiveness.