Coding

Part:BBa_K4229013

Designed by: Nikita Edel   Group: iGEM22_Freiburg   (2022-09-28)
Revision as of 23:11, 11 October 2022 by NGensch (Talk | contribs)


TnaA with N-terminal SnoopCatcher

TnaA belongs to the family of tryptophanase (tna) operon leader peptide. Tryptophanase catalyses the degradation of L-tryptophan to indole, pyruvate and ammonia, enabling the bacteria to utilise tryptophan as a source of carbon, nitrogen and energy. The tna operon of E. coli contains two major structural genes, tnaA and tnaB.

TnaA codes for a tryptophanase. This tryptophanase turns L-tryptophan into either Indol. This biobrick consists of a version of TnaA, which is fused with the snoopcatcher(BBa_K4229009) on the N-terminal of the protein. With that, we made it able to be recruted into the wiffelball we used in our project.


Grafik indigo weier bg.png

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 [2].


References
[1]. A transcriptional pause synchronizes translation with transcription in the tryptophanase operon leader region. Gong F, Yanofsky C. J. Bacteriol. 185, 6472-6, (2003). View articlePMID: 14563884
[2] 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 977
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 977
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 977
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 977
    Illegal AgeI site found at 142
    Illegal AgeI site found at 228
    Illegal AgeI site found at 1600
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None