Difference between revisions of "Part:BBa K4378000:Design"

 
 
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===Design Notes===
 
===Design Notes===
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For potential in vitro use of the enzyme an T7-Tag is attached to allow purification through affinity chromatography.
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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  
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+
  
  
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===References===
 
===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.
 +
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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.

Latest revision as of 14:19, 28 September 2022


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.