Composite

Part:BBa_M50437:Design

Designed by: Lillian Zhu, Kelsie Wysong, Eva Frankel   Group: Stanford BIOE44 - S11   (2018-04-25)
Revision as of 00:01, 12 June 2018 by Evaf (Talk | contribs)

2,3-DHB Biosynthesis Construct, with entC and ahpC


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 234
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 234
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 234
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 234
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 234
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part contains an IPTG-inducible T5 promoter (Part:BBa_M50075) which was unmodified. By saturating cell media with IPTG, the T5 promoter can be fully induced in order to make an excess of ehpC and entC. This part also contains two strong ribosome binding sites: one before ahpC, and another before entC. The same strong RBS (part:BBa_M50080) was used twice, and it was used unmodified. We found the ahpC amino acid sequence on Uniprot and optimized the codon sequence for E. coli using the Integrated Data Technologies Codon Optimization Tool (IDT CodonOpt). We modified this sequence by adding FLAG (Part:BBa_T2004) onto the end of our ahpC sequence. This modified ahpC sequence is part BBa_M50435. Similarly, we acquired the entC amino acid sequence from Uniprot and optimized the codon sequence for E. coli using IDT CodonOpt. We modified this by adding a 6xHis tag (Part:BBa_K112703) to the C-terminus. This modified entC sequence is part BBa_m50436 in the iGEM registry. Finally, we added the T7 Phage terminator downstream of our 6xHis tag (Part:BBa_M50060). This is a strong terminator for bacterial expression.

Source

T5 promoter: BBa_M50075
Strong RBS: BBa_M50080
entC: BBa_M50436
ahpC: BBa_M50435
T7 terminator: BBa_M50080

References