Part:BBa_K2984050

L1c-PsaDintron-bleRscp-ARS-MHETase-SP20-RbcS2

This vector is a part of the Chlamy-HUB-Collection. This part is composed of the PsaD promoter with an expression enhancing intron PsaDintron, the bleomycin resistance with self cleaving peptide scp, the secretion signal ARS, the MHETase enzyme, kindly provided by the TU Kaiserslautern 2019 team, the secretion enhancing SP20 glycomodule, and the Rbcs2 terminator. The part can be used to express and secrete the MHETase enzyme in C. reinhardtii. The bleomycin resistance leads to a higher expression of the enzyme, due to its working mechanism. For every two bleomycin molecules, the bleomycin resistance gene must be expressed once. The resistance works in a ratio of 1:2 to the antibiotic (Hayes et al., 1990). By having the resistance in the same open reading frame, the enzyme is also expressed, leading to a higher expression of the enzyme (Lumbreras et al. 1998).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2614
  Illegal PstI site found at 2938
  Illegal PstI site found at 3281
  Illegal PstI site found at 4091
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2614
  Illegal PstI site found at 2938
  Illegal PstI site found at 3281
  Illegal PstI site found at 4091
  Illegal NotI site found at 2949
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3859
  Illegal BamHI site found at 4
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2614
  Illegal PstI site found at 2938
  Illegal PstI site found at 3281
  Illegal PstI site found at 4091
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2614
  Illegal PstI site found at 2938
  Illegal PstI site found at 3281
  Illegal PstI site found at 4091
  Illegal NgoMIV site found at 1607
  Illegal NgoMIV site found at 2547
  Illegal NgoMIV site found at 3008
  Illegal NgoMIV site found at 3038
  Illegal NgoMIV site found at 3353
  Illegal NgoMIV site found at 3371
  Illegal NgoMIV site found at 3407
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Fig.1 - Image of a successful transformation in E. coli after ligation of the construct. The construct can then be isolated from E. coli to be transformed in C. reinhardtii.

References

1. Lumbreras, V., Stevens, D. R., & Purton, S. (1998). Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. The Plant Journal, 14(4), 441-447.
2. Hayes, J. D., & Wolf, C. R. (1990). Molecular mechanisms of drug resistance. Biochemical Journal, 272(2), 281.