Part:BBa_K4229016

RBS-snoopCatcherTnaA-RBS-FRE

This composite part was used to for the coexpression of two target genes of the indigo/indirubin pathways. The first one is TnaA with N-terminal SnoopCatcher (BBa_K4229013) and the second one being Fre (BBa_K4229001). These two genes build together with the BBa_K4229018 all enzymes of the indigo/indirubin pathway.

Figure 1: Schematic representation of the indigo/indirubin pathway. L-tryptophan is imported by the membrane protein TnaB (low affinity tryptophan permease). L-tryptophan is cleaved into indole, NH4+ and pyruvate by the tryptophanase TnaA. The reaction continues by the hydroxylation of indole through XiaI. To enhance the effectivity of this enzyme, the NAD(P)H-flavin reductase provides XiaI with FADH2 by adding hydrogen to FAD. Finally, indole is transformed to either 3-Hydroxyindole or 2-Hydroxyindole. These two substances spontaneously react to 3-Oxindole and 2-Oxindole through the secession of hydrogen from the OH-group. Through spontaneous dimerization indigo and indirubin are formed. Graphic adapted from [1].

Usage and Biology

The production was tested under the T7 promoter and LacI operator in those two Biobricks: BBa_K4229033 and BBa_K4229034)

References
[1] H. Yin et al., “Efficient Bioproduction of Indigo and Indirubin by Optimizing a Novel Terpenoid Cyclase XiaI in Escherichia coli,” ACS Omega, vol. 6, no. 31, pp. 20569–20576, 2021, doi: 10.1021/acsomega.1c02679.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 990
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 990
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 990
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 990
  Illegal AgeI site found at 155
  Illegal AgeI site found at 241
  Illegal AgeI site found at 1613
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2150
  Illegal SapI.rc site found at 2270