Difference between revisions of "Part:BBa K4656007"

Revision as of 01:53, 8 September 2023

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

1.Western blotting results:

2.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

@@ Line 10: / Line 10: @@
 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==
-Western blotting results:
+.Western blotting results:
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-gray-value evaluation:
+.gray-value evaluation:
 <html><BR><BR><center><img style="display: block;-webkit-user-select: none;margin: auto;background-color: hsl(0,0%,90%);transition: background-color 300ms;" src="https://static.igem.wiki/teams/4656/wiki/wb.png"width="250" height="120"></center>
 <html><BR><BR><center><img style="display: block;-webkit-user-select: none;margin: auto;background-color: hsl(0,0%,90%);transition: background-color 300ms;" src="https://static.igem.wiki/teams/4656/wiki/gray-value.png"width="392" height="292"></center>