Part:BBa_K3075002

Introduction

TycA-S563A-SpyT consists of the enzyme Tyrocidine synthase 1 fused at the C-terminus to a short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. Our team utilised a sequence originating from Brevibacillus parabrevis with a S563A amino acid mutation. (1) (2)

Usage and Biology

Tyrocidine Synthase 1 (TycA) is known to be involved in tyrocidine biosynthesis, a part of antibiotic biosynthesis. In the biosynthesis of the Paclitaxel side chain, TycA combines the beta-phenylalanine and the CoA thioester, and can also catalyse the production of phenylisoserinyl-CoA. (3) Recombinant TycA has a sequence of 1088 amino acid residues with a molecular mass of 122,672.

Characterisation

The TycA-S563A-SpyT gBlock was synthesised by IDT. TycA-S563A-SpyT was ligated into pET19b plasmid backbone by Gibson assembly and transformed into competent T7 express E.coli cells. Colony PCR was performed using primers listed below and the amplicon was visualised by gel electrophoresis.

Primers used:

- T7 Forward : 5’-TAATACGACTCACTATAGGG

- T7 Reverse : 5’-GCTAGTTATTGCTCAGCGG

A small scale grow up of colonies was performed and plasmid DNA was extracted via a QIAGEN miniprep kit. Miniprepped samples were visualised via gel electrophoresis (Figure 1) and submitted for sequencing by the Ramaciotti Centre for Genomics (Figure 2).

Starter culture was made for colonies of interest and large-scale grow up was done. Expression of the recombinant protein was induced using IPTG and harvested cells were lysed via Sonication. The His-tagged protein was then purified via IMAC. Results are shown on figure 3.