Part:BBa_K3075002

TycA^S563A-SpyT-His

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2289
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2299
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1223
  Illegal BsaI.rc site found at 2143
  Illegal SapI.rc site found at 2499
  

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)

The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.

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

TycA^S563A-SpyT gene inserts were obtained in two fragments (TycA-1 and TycA-2), with a 60 bp overlap between the two fragments. Ligation of both TycA-1 and TycA-2 fragments with pET-19b backbone (provided by Dr Dominic Glover) was attempted via Gibson Assembly. The assembly mix was incubated for 1 hour at 37°C before transforming into T7 Express E. coli cells by heatshocking at 42°C. Transformed cells were plated on an LB Agar plate supplemented with Ampicillin at working concentration for selection, and incubated overnight at 37°C. Transformant colonies were screened for recombinant plasmids by colony PCR under the following conditions: Initial denaturation at 97°C for 3 minutes, 30X cycles of: denaturation at 97°C for 10 seconds, annealing at 67.6°C for 30 seconds and extension at 72°C for 50 seconds. Final extension at 72°C for 5 minutes. PCR products were run on a 1% agarose gel in 1X TAE Buffer for 1 hour at 100 V. Colony PCR performed on the TycA colonies showed bands which did not align with the theoretical length of 3.383 kb. Sanger sequencing results confirmed that the colonies submitted did not contain the desired TycA gene insert (Figure omitted).

As TycA^S563A-SpyT is a larger gene fragment (3.3kb), we attempted to increase the incubation time during Gibson Assembly to allow for enough time for the complementary overhangs to properly anneal between the insert and vector.

The following conditions have been attempted:

• Gibson Assembly incubation – 15 minutes, 60 minutes.
• Gibson master mix: commercial compared with home-made.
• Insert to vector: 3X excess, 5X excess.

The 60 minutes (Figure 6A) incubation produced greater number of colonies compared to the 15 minutes (Figure 6B). This suggests the longer incubation time allows for the ligation of fragments into a functional circularised plasmid. Colony PCR results of these colonies revealed __________. The commercial master mix had more colonies than the home-made, but not significantly (Figure 6C).

With multiple repetitions of the Gibson Assembly procedure, the lack of successful recombinant plasmids raises the question, that perhaps the gene fragments are not correctly synthesised. We plan to confirm the sequence of our gene fragments empirically, by Sanger sequencing.