Part:BBa_K2148004

Homology-psaA promoter/5UTR/NTAG

A level-0 Phytobrick coded as a promoter/5UTR with a Chlamydomonas reinhardtii chloroplast homology region upstream of the promoter sequence to allow for homologous recombination. The homology region is left from the trnE2-psbH integenic region in the Chlamydomonas reinhardtii chloroplast obtained from Saul Purton's backbone pSRSapI. This region is used commonly by experts in the field due to high yields of gene of interest.

Usage and Biology

This part can be used with another level-0 Phytobrick to create a transcriptional unit, accelerates the assembly process as the two parts are together already. Have included the phytobrick standard of an NTAG as there was no "appriopriate" site for such a phytobrick.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4
  Illegal XbaI site found at 50
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4
  Illegal XbaI site found at 50
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4
  Illegal XbaI site found at 50
  
• 1000
  COMPATIBLE WITH RFC[1000]

Verification

Part was verified through sequencing. Data not shown due to part size.

The functionally of the part was also confirmed when a construct, containing it, aadA CDS and rbcL 3UTR/TERM successfully conferred resistance to spectinomyin, when transfected into the chloroplasts of Chlamydomonas reinhardtii.

References

Wannathong T, Waterhouse JC, Young REB, Economou CK, Purton S. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Applied Microbiology and Biotechnology. 2016;100:5467-5477. doi:10.1007/s00253-016-7354-6.