Part:BBa_K4179013

ChimeraI under Rhlr promoter + P2A + mCherry reporter

This composite part comprises of an umbelliferone-6 prenyl transferase ChimeraI (BBa_K4179011), which was engineered by the team of Technion 2022-Israel, under the rhlr-regulated promoter (BBa_R0071). Downstream to ChimeraI 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 547
  Illegal PstI site found at 1792
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1357
  Illegal PstI site found at 547
  Illegal PstI site found at 1792
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 2152
  Illegal PstI site found at 547
  Illegal PstI site found at 1792
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 2152
  Illegal PstI site found at 547
  Illegal PstI site found at 1792
  Illegal NgoMIV site found at 962
  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 ChimeraI, 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 ChimeraI's expression.

ChimeraI 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 ChimeraI enzyme, and its construction process, visit its page in the registry (BBa_K4179011), 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.