Part:BBa_K4144008

This part is designed to express S-adenosylmethionine synthetase opSam2 to synthesize SAMe

Results

This part is designed to express SAMe synthetase opSam2 derived from Sam2 in Saccharomyces cerevisia mitochondron. S. cerevisiae was reported to possess two kinds of SAM synthetase, SAM1 and SAM2, in which the latter is insensitive to the product inhibition of SAM synthesis. This property is the reason why we chose it as the engineered enzyme to synthesize SAMe. It can synthesize SAMe from L-Methionine and ATP (Fig. 1). Our design is to put such part downstream of one promoter of oscillator, so we need to verify its induction expression and its enzymatic ability.

Figure. 1 Chemical Reaction of SAMe synthesis catalysed by SAM synthetase Sam2.

We performed expression verification through western blot (Fig. 2). As the result shows, we can figure out that the band between 40 and 55 displays the existence of 6xHis::opSam2 whose molecular weight can reach 45.80 kDa. As the induction during increases, the band light becomes stronger, which means that the expression of 6xHis::opSam2 is increased. Meanwhile, we may find some shifting phenomenon in rear lanes like lane 6, 7 and 8. This could be caused by the degradation of opSam2 due to the much longer induction.
Then we test its enzymatic ability through SAMe quantification kit (Fig. 3). We have successfully verified its enzymatic function.

Figure. 2 Enzymatic Function Verification of opSam2 in B. subtilis.

Figure. 3 Enzymatic Function Verification of opSam2 in B. subtilis.

Reference

[1]Kefala G, Kwiatkowski W, Esquivies L, Maslennikov I, Choe S. Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from Escherichia coli. J Struct Funct Genomics. 2007 Dec;8(4):167-72. doi: 10.1007/s10969-007-9033-4. Epub 2007 Nov 6. PMID: 17985211.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 356
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 340