Part:BBa_K4656007

Plam-TPH-TDC

Under the activation of Plam, downstream genes Tph1 and Tdc1 are expressed. The Tph gene encodes tryptophan hydroxylase (TPH) and the Tdc gene encodes tryptophan decarboxylase (TDC). These two enzymes play a vital 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_R0051, BBa_K4656002 and BBa_K4656006, we designed a gene route Plam-TPH1-TDC1-EGFP, then we 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

We introduced the plasmid pet28a-plam-TDC-TPH into the receptive E. coli. The SDS-PAGE results showed that TDC and TPH were highly expressed by the engineered E. coli (Figure 1a,1b,1c), while the growth was unaffected compared to that of the empty-loading E. coli (Figure 1d).

Figure1.TPH and TDC can be expressed by engineered bacteria and exert no effect on their proliferation.

a.Original image of SDS-PAGE presenting the TPH and TDC expression, beat-actin as a control

b.Grayscale stripe extraction from the original image of SDS-PAGE

c.Histogram of grayscale values presenting the TPH and TDC expression

d.OD evaluation of the control and engineered bacteria throughout overnight culture

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