Part:BBa_K2906012

T7 promoter- RBS- Sam8- T7 terminator

sam8 gene of Saccharothrix espanaensis encodes a tyrosine ammonia lyase (TAL) which converts L-tyrosine to p-Coumaric acid. It is the first enzyme required in the vanillin biosynthesis pathway. This gene was codon optimised by GeneArt GeneOptimiser for expression in E. coli. A His-tag and a TEV protease cleavage site were added to it to allow functional analysis of the protein. The T7 promoter, RBS and T7 terminator were fused after cloning into pETM-11 plasmid.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 537
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 421
  Illegal AgeI site found at 637
  Illegal AgeI site found at 1248
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation:

To show the functionality of the gene after cloning into pETM-11, we performed SDS-PAGE and Western blot to prove expression of the protein TAL (Figure 1).

Figure 1. SDS-gel (a) and western blot (b) of TAL, C3H and COMT, the first three enzymes in the vaniliin biosynthesis pathway encoded by sam8, sam5 and comt. The expected sizes of the proteins are 57.4 kDa, 60.49 kDa and 41.39 kDa for TAL, C3H and COMT respectively. The negative control (-ve) is the pETM-11 (empty vector) and the positive control (+ve) is the pETM-11-ABU58587 expressing a 36 kDa His-tagged protein.

We also detected and quantified the product, p-coumaric acid, from a BL21 (DE3) bacterial culture carrying the pETM-11-sam8 plasmid by UPLC-MS/MS (Figure 2). A 10 mL culture (1/100) of the respective strain in 2xYT media, which contains tyrosine, was induced by 100 µM IPTG and grown for 72 hours at 26°C and 180 rpm which was then processed for analysis by UPLC-MS/MS; 213.954 µM of p-coumaric acid was detected.

Figure 2. UPLC–MS/MS chromatograms of p-coumaric acid (a), the product of TAL, from BL21 (DE3) bacterial culture carrying pETM-11-sam8 (b) grown in 2xYT media which contains the precursor tyrosine. X-axis show retention time.

References:

Gruz, J., Novák, O. and Strnad, M. (2008). Rapid analysis of phenolic acids in beverages by UPLC–MS/MS. Food chemistry, 111(3), pp.789-794. DOI: https://doi.org/10.1016/j.foodchem.2008.05.014.