Composite

Part:BBa_K4179014

Designed by: Yasmin Habib   Group: iGEM22_Technion-Israel   (2022-10-05)


ChimeraII under Rhlr promoter + P2A + mCherry reporter

This composite part comprises of an umbelliferone-6 prenyl transferase ChimeraII (BBa_K4179012), which was engineered by the team of Technion 2022-Israel, under the rhlr-regulated promoter (BBa_R0071). Downstream to ChimeraII there is a P2A sequence (BBa_K4179005), which is a self-cleavage element that induces ribosomal skipping during translation of a protein inside the cell. Downstream to which there is an mCherry (BBa_K106005) reporter gene, and an rrnB terminator sequence (BBa_K4047025).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 2152
    Illegal PstI site found at 778
    Illegal PstI site found at 1792
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1357
    Illegal PstI site found at 778
    Illegal PstI site found at 1792
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 843
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 2152
    Illegal PstI site found at 778
    Illegal PstI site found at 1792
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 2152
    Illegal PstI site found at 778
    Illegal PstI site found at 1792
    Illegal NgoMIV site found at 883
    Illegal AgeI site found at 1431
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

In this genetic system, the mCherry expression is tied directly to the start codon of ChimeraII, while not being covalently bound to it. In such a design the mCherry signal corresponds to a 1:1 ratio, at the very least, which allows for a lower estimate of ChimeraII's expression.

ChimeraII enzyme is the first enzyme in the decursin biosynthesis pathway, which the team was attempting to clone into a bacterial system. For more information about the ChimeraII enzyme, and its construction process, visit its page in the registry (BBa_K4179012), or visit the team’s wiki page.


The team used this sequence in the rhlr-tdpp7-m/cherry plasmid, which already holds the rhlr gene.

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