Difference between revisions of "Part:BBa K2043007:Design"

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===Design Notes===
 
===Design Notes===
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bpul is compatible with both pCOLA vector and with the PhytoBrick format. IDT’s Codon Optimization Tool (https://eu.idtdna.com/CodonOpt) was used to codon optimize the sequence of the bpul gene for expression in E.coli. The sequence was checked and corrected for potential BpiI, BsaI and BsmBI recognition sites. These sites cannot be present in the coding sequence, because the BpiI, BsaI and BsmBI enzymes are widely used in phytobrick cloning (http://2016.igem.org/Resources/Plant_Synthetic_Biology/PhytoBricks).  
+
IDT’s Codon Optimization Tool (https://eu.idtdna.com/CodonOpt) was used to codon optimize the sequence of the bpul gene for expression in E.coli. The sequence was checked and corrected for potential BpiI, BsaI and BsmBI recognition sites. These sites cannot be present in the coding sequence, because the BpiI, and BsaI enzymes are widely used in phytobrick cloning (http://2016.igem.org/Resources/Plant_Synthetic_Biology/PhytoBricks).  
 
BsaI recognition site was attached to the 5'-end of the gene sequence. This will allow cloning the the part in the Phytobrick. To the 3’-end the His-tag and a BsaI recognition site were attached. This will allow us to purify the protein and also fit the part in the Phytobrick.
 
BsaI recognition site was attached to the 5'-end of the gene sequence. This will allow cloning the the part in the Phytobrick. To the 3’-end the His-tag and a BsaI recognition site were attached. This will allow us to purify the protein and also fit the part in the Phytobrick.
  

Latest revision as of 02:06, 27 October 2016


bpuI laccase codon optimized for E. coli


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 888
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 888
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 888
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 888
    Illegal AgeI site found at 52
    Illegal AgeI site found at 247
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

IDT’s Codon Optimization Tool (https://eu.idtdna.com/CodonOpt) was used to codon optimize the sequence of the bpul gene for expression in E.coli. The sequence was checked and corrected for potential BpiI, BsaI and BsmBI recognition sites. These sites cannot be present in the coding sequence, because the BpiI, and BsaI enzymes are widely used in phytobrick cloning (http://2016.igem.org/Resources/Plant_Synthetic_Biology/PhytoBricks). BsaI recognition site was attached to the 5'-end of the gene sequence. This will allow cloning the the part in the Phytobrick. To the 3’-end the His-tag and a BsaI recognition site were attached. This will allow us to purify the protein and also fit the part in the Phytobrick.

Source

Bacilus pumillus

References

Reiss R., Ihssen J., Thöny-Meyer L. (2011). Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC Biotechnology 11:9