Part:BBa_K4378000:Design

TphB: An effective overexpression casette

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 288
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 409
  

Design Notes

Coding sequence for tphB enzyme was taken from Uniprot: Q3C1E1 and nucleotide sequence was codon-optimised for expression in E. coli. BioBrick prefix, containing restriction sites for EcoRI and XbaI, and suffix with sites for restriction by SpeI and PstI were added on ends of the construct for easier insertion into vectors. Our construct was also designed with RBS, T7 promoter and terminator and lac operator for optimal induction of protein expression (Shepherd et al. 2017, Du et al. 2012) and C-terminal histidine tag for enzyme purification on immobilized metal affinity chromatography

Source

https://www.uniprot.org/uniprotkb/Q3C1E1/entry

References

Shepherd TR, Du L, Liljeruhm J, Samudyata null, Wang J, Sjödin MOD, Wetterhall M, Yomo T, Forster AC. 2017. De novo design and synthesis of a 30-cistron translation-factor module. Nucleic Acids Research 45: 10895–10905.

Du L, Villarreal S, Forster AC. 2012. Multigene expression in vivo: supremacy of large versus small terminators for T7 RNA polymerase. Biotechnology and Bioengineering 109: 1043–1050.