Part:BBa_K3077200

Expression of lipIAF5-2 under constitutive promoter

The part BBa_K3077200 is the composite part of the new heterologous lipase(lipIAF5-2). The genomic sequence of the lipIAF5-2 gene is referenced from ENA (Gene ID EU660533). The lipIAF5-2 gene, registered as BBa_K3077202, is designed to be the composite part by adding a constitutive promoter BBa_J23111, strong RBS BBa_B0034 and double terminator BBa_B0015.

• J23111 is a constitutive promoter that allows continual expression of the gene, it is a consensus promoter sequence and the strongest member of the family, so it can promote a high rate of gene expression.
• B0034, a strong RBS, to achieve a higher expression level.
• B0015 is a double terminator combining B0010 and B0012, and is one of the most commonly used and reliable terminator in the iGEM catalog.

The gene BBa_K3077202 is a new heterologous lipase identified by a metagenome library encoded as lipIAF5-2. It is a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa[1]. It is a newly discovered novel gene sequence that showed no more than 52% identity with other lipases[1]. The lipase showed higher activity with long-length acyl chains, showing maximal activity with p-NPM (C14) and about 70% with p-NPM (C16) and p-NPM (C18)[1]. It is extracellular and is secreted out of the cell[1]. Being a true lipase, it is able to efficiently synthesize short chain esters which produces sweet-smelling aromas by transesterification and esterification reactions in organic media. It showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. [2]

This plasmid containing composite part BBa_K3077200 is ordered and synthesized by IDT. The plasmid is transformed in E.coli. However, we don’t have sufficient time to verify the E.coli with Colony PCR as planned.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 497
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 497
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 497
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 497
  Illegal NgoMIV site found at 323
  
• 1000
  COMPATIBLE WITH RFC[1000]