Part:BBa_K3407016:Design

B. subtilis decarboxylase (bsdBCD) with inducible T7 promoter, RBS and T7 terminator

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 NgoMIV site found at 531
  Illegal AgeI site found at 121
  Illegal AgeI site found at 373
  Illegal AgeI site found at 1202
  Illegal AgeI site found at 1292
  Illegal AgeI site found at 1494
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design notes

The sequence of the three genes (bsdBCD) was taken from Bacillus subtilis subsp. subtilis str. 168 complete genome (GenBank: AL009126.3), 412540...414822. The genes were codon-optimized for expression in Escherichia coli using the GenSmartTM Codon Optimization Tool. The forbidden restriction enzymes sites are the following: BioBrick forbidden restriction sites (EcoRI, XbaI, SpeI, PstI) and Type IIS forbidden restriction sites (BsaI, SapI and Bpil). As the three genes were not in the same reading frame due to nucleotides in between them, each of them was codon-optimized separately.

The expression cassette of this part was synthesized in the backbone vector pTWIST_Amp_HighCopy from Twist bioscience, containing the viral promoter and terminator from the T7 bacteriophage. This synthetically synthetised plasmid is referred as pTWIST_bsdBCD.

This part was designed as if it was cloned using Modular Cloning Toolkit [1][2], so it contians the scars left after assembly.

Source

Synthetically synthesised.

References

1. Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS one, 6(2), e16765.
2. Werner, S., Engler, C., Weber, E., Gruetzner, R., & Marillonnet, S. (2012). Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioengineered bugs, 3(1), 38–43.