Part:BBa_K2984048

L1c-PsaD-bleRscp-ARS-PETase-SP20-RbcS2

This vector is a part of the Chlamy-HUB-Collection. This part is composed of the PsaD promoter, bleomycin resistance with self cleaving peptide scp, the ARS secretion signal, the PETase enzyme, the secretion enhancing SP20 glycomodule, and the Rbcs2 terminator. The part can be used to express and secrete the PETase 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
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2249
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4
  Illegal XhoI site found at 1973
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1607
  
• 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.