Composite

Part:BBa_K4179003

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


PcPT under Rhlr promoter + P2A + mCherry reporter

This composite part comprises of an umbelliferone-6 prenyl transferase PcPT (BBa_K4179000), under the rhlr-regulated promoter (BBa_R0071). Downstream to PcPT 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 EcoRI site found at 271
    Illegal EcoRI site found at 703
    Illegal EcoRI site found at 1066
    Illegal XbaI site found at 2146
    Illegal PstI site found at 226
    Illegal PstI site found at 589
    Illegal PstI site found at 1786
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 271
    Illegal EcoRI site found at 703
    Illegal EcoRI site found at 1066
    Illegal NheI site found at 1351
    Illegal PstI site found at 226
    Illegal PstI site found at 589
    Illegal PstI site found at 1786
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 271
    Illegal EcoRI site found at 703
    Illegal EcoRI site found at 1066
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 271
    Illegal EcoRI site found at 703
    Illegal EcoRI site found at 1066
    Illegal XbaI site found at 2146
    Illegal PstI site found at 226
    Illegal PstI site found at 589
    Illegal PstI site found at 1786
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 271
    Illegal EcoRI site found at 703
    Illegal EcoRI site found at 1066
    Illegal XbaI site found at 2146
    Illegal PstI site found at 226
    Illegal PstI site found at 589
    Illegal PstI site found at 1786
    Illegal AgeI site found at 1425
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

In this genetic system, the mCherry expression is tied directly to the start codon of PcPT, 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 PcPT's expression.

PcPT enzyme is the first enzyme in the decursin biosynthesis pathway, which the team of Technion 2022 was attempting to clone into a bacterial system. For more information about the PcPT enzyme, visit its page in the registry (BBa_K4179000), 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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Categories
Parameters
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