Team:KCL UK/CompositeParts

Renervate

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Composite Parts

Welcome to our Composite Parts library

Here is a closer look into our composite parts and how they will function as a co-expression system:

Renervate Plasmid Backbone

Using the T7 promoter we will control transcription of both plasmids under expression of T7 polymerase. This will be induced by IPTG. The lac operator sequence will not only be present on the lac cassette in the host’s genome but also downstream of the ribosome binding site to prevent any background expression. Ampicillin resistance has been chosen as a genetic marker for transformed colonies. We have replaced the pMB1 replicon with Bluescript II SK +f1 ori to reduce toxicity to the host strain as well as maintain adequate levels of the vector. A 7xHis tag has been added at the 3' end of sequence to allow for his tag purification of the product. This part is called BBa_K3635008.

Pvfp-5β Expression Vector:

The T7 promoter has been used to control expression under T7 polymerase. This will allow us to induce transcription with IPTG as our host E.coli will contain the lac cassette in its genome.We have further added the lac operator sequence downstream of the RBS to eliminate any background transcription. A His Tag has been added to give 7 histidines at the C terminus of the protein to allow for purification.

Tyrosinase Expression Vector

The T7 promoter has been used for the timely induction of both expression vectors to ensure in vivo polymerisation occurs parallel to protein production. It is essential that both plasmids in the co-expression system are working under the same promoter as if Pvfp-5β is expressed into the cytoplasm with no tyrosinase present, tyrosine residues will be converted to dopamine. These dopamine molecules will then undergo irreversible spontaneous oxidation which will severely limit adhesion. This tyrosinase enzyme must be expressed in ORF438 to ensure that copper atoms can incorporate at the active site, This will give optimum enzyme activity.