Composite

Part:BBa_K4656007

Designed by: Xinji Gu   Group: iGEM23_NMU-China   (2023-08-09)
Revision as of 01:51, 8 September 2023 by Juejue (Talk | contribs)


Plam-TPH-TDC

Under the activation of Plam, downstream genes TPH1 and TDC1 are expressed. The Tph1 gene encodes tryptophan hydroxylase (TPH) and the Tdc1 gene encodes tryptophan decarboxylase (TDC). These two enzymes play a huge role in the production of 5-HT. Tryptophan decarboxylase (TDC) is the key enzyme, which can catalyze the conversion of tryptophan (an essential amino acid) to 5-hydroxytryptophan (5-HTP). Tryptophan hydroxylase (TDC) can remove the carboxyl group of 5-hydroxytryptophan (5-HTP) and convert it into serotonin (5-hydroxytryptophan, 5-HT) molecules. Serotonin can play a role in promoting intestinal motility, which is beneficial for improving constipation. Notably, promoter Plam can be inhibited by binding to CI repressor proteins.

Usage and Biology

To verify the feasibility of the metabolic module pathway, that is, the pathway for the production of sufficient tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC) in engineered bacteria. Using components BBa_K4656001, BBa_K4656002 and BBa_K4656006, we designed a gene route Plam-TPH1-TDC1-EGFP, cloned it into the target vector pET28a(+), and then transformed the engineered bacteria. The routes formed by these components can be used to verify the viability of metabolic modules. With untreated pET28a as the control group, we conducted experiments such as WB, fluorescence intensity (OD600) determination, gray value analysis, etc., to verify the feasibility of the metabolic module pathway.

Experimental results



gray-value evaluation:



3.The production of enzymes exerts no effect on bacterial growth:


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 959
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2641
    Illegal BamHI site found at 2125
    Illegal BamHI site found at 2227
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1733
    Illegal NgoMIV site found at 2168
    Illegal NgoMIV site found at 2723
    Illegal AgeI site found at 734
    Illegal AgeI site found at 1871
    Illegal AgeI site found at 2795
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 301


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