Difference between revisions of "Part:BBa K4229016"
 
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<partinfo>BBa_K4229016 short</partinfo>
 
<partinfo>BBa_K4229016 short</partinfo>
  
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These are the genes we put inside MCS1 of our vector pCDF-Duet-1. The first gene is the fusion protein of TnaA with the snoopcatcher(BBa_K4229013) on the C-terminal, the second one being Fre(BBa_K4229001). It was decided to put the enzymes with the catcher at the beginning of each MCS as those are our most important proteins, which we wanted to have produced the most. These two genes build together with the Biobrick BBa_K42218 all enzymes of the indigo/indirubin pathway. The production was tested under the T7 promotor and LacI operator in those two Biobricks: BBa_K4229033 and BBa_K4229034)
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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.  
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[[File:Grafik indigo weier bg.png|900px|thumb|left|
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<b>Figure 1: Schematic representation of the indigo/indirubin pathway.</b> 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].]]
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[[File:Grafik indigo weier bg.png|900px|thumb|left|]]
 
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===Usage and Biology===
 
===Usage and Biology===
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The production was tested under the T7 promoter and LacI operator in those two Biobricks: BBa_K4229033 and BBa_K4229034)
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<b>References</b>
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[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.
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Latest revision as of 23:51, 11 October 2022


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