Part:BBa_K2997000

Tyrosine transporter (tyrP)

Background

It’s the gene encode for tyrosine transporter that is involved in transporting tyrosine across the cytoplasmic membrane. Hence, we used PCR to amplify this gene from E. coli MG1655 and incorporated it to our E. coli, allowing it to take in more tyrosine.

Expression in E. coli

We amplified the tyrP gene and its promoter from E. coli MG1655 and cloning into pSB4A3. Then transformed the plasmid into DH5a and E. coli Nissle 1917, and extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful.

TAL and TyrP Functional Assay

We performed a functional test using n-octanol extraction method (https://2019.igem.org/Team:NCKU_Tainan/Protocols) , and with this assay, we can further improve the conversion of tyrosine into p-Coumaric acid by adding a tyrosine transporter (BBa_K2997000). As seen in figure., when BBa_K2997000 is added, the production of p-Coumaric acid is significantly higher than when BBa_K2997000 is not added. When tyrosine transporter is introduced into E. coli Nissle containing BBa_K2997009 and BBa_K2997010, conversion of tyrosine into p-Coumaric acid is increased by 1.44-fold and 1.31-fold respectively.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 278
  
• 1000
  COMPATIBLE WITH RFC[1000]