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Creation of the Taq Polymerase Part

Making the Inducible T7 Expression Vector

The aim for this section is to create a an inducible expression backbone. This will be achieved by altering the backbone biobrick BBa_J04500, by removing the -35 and -10 elements (promoter) and inserting the bacteriophage T7 promoter. the strategy used to will be Quikchange XL mutagenesis to alter the promoter elements into restriction sites:

• -35 --> AflII
• -10 --> BamH1

These restriction sites are NOT found in the BBa_J04500 part or vector (pSB1A2), so there will be no cross cutting.

By the T7 promoter is small enough for it to be constructed from primers. Two ligation ends would be designed to match the sticky ends of the restriction sites introduced. The T7 can thus be inserted as the promoter of the system, being regulated by IPTG (LacO site) and glucse/cAMP (CAP-binding site).

Results