Part:BBa_K2276001

T7 promotor and the coding sequneces of tryptophan decarboxylase (TDC) with N-terminal His-tag

Biobrick BBa_K2276001 is a composite, consisting of T7 promotor,6xHis tag, thrombin site,and the coding sequence of tryptophan decarboxylase (TDC).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1015
  Illegal BamHI site found at 1682
  Illegal XhoI site found at 1674
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 639
  Illegal AgeI site found at 1272
  Illegal AgeI site found at 1521
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Tryptophan decarboxylase (TDC) can convert 5-hydroxytrypthphan into serotonin. In animals, serotonin is synthesized in two enzymatic steps. In the first step, the essential amino acid tryptophan is hydroxylated to 5-hydroxytryptophan by tryptophan hydroxylase, followed by a decarboxylation step in which tryptophan decarboxylase (TDC) convert 5-hydroxytrypthphan into serotonin [1]. Similarly, in plants, serotonin biosynthesis requires two enzymes. However, the first reaction is catalyzed by TDC, converting tryptophan into tryptamine. For easily detection the products, we chose the metabolic pathway of plants to be a guidance. Actually, the enzyme TDC exists in many species, but we should choose one with high bioactivity in E.coli system to make the final product——melatonin arrive at the maximum expression level. So, The TDC we used comes from the plants genome —— Catharanthus roseus , showing the higher bioactivity in an E.coli system than other resource[2]. What’s more, in order to introduce it into the E.coli system, we also did the codon optimisation.

Serotonin acts as a neurotransmitter in animals, which is primarily found in the central nervous system, and blood platelets. It is generally thought to contribute to the regulation of mood, appetite and sleep. Thus, the TDC enzyme plays an important role in the pathway.

Results

Link the TDC with N-terminal His tag:

In our experiments, we want to figure out and confirm the function of TDC. In other words, only we obtained the complete pure enzymes, we could find out its function. Thus, firstly we link TDC with the vector pET15(+), which has N-terminal His tag that make the protein purification easier. And this work was verified by bacterial colony PCR (Figure.1).

Confirm the TDC was expressed successfully in E.coli BL21( DEC):

When the plasmid was constructed successfully, we must detect whether the enzyme we obtain can express in E.coli system. Therefore, we transferred the pET15-TDC plasmid into E.coli BL21 (DEC) strain [3] for expression. What’s more, in order to find out where the protein exactly exists, we used the SDS-PAGE to detect the different compositions of the bacteria lysate (Figure.2).

Prove the TDC really function in E.coli system:

However, there was still a doubt that if the enzyme really works in the E.coli BL21 (DE3) system. Hence, to verify the TDC enzyme catalyzes the reaction to convert tryptophan into tryptamine, we used the HPLC (High Performance Liquid Chromatography), a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture, to test the reduction of the substrate and the increment of the product. We did this by two ways: in vivo, we added the substrate tryptophan into the culture media. After a period of culturing, we tested the tryptophan content in the media and the product content in bacterial lysate (Figure.3). For another thing, in vitro, we lysed the bacteria at first, and then added the substrate and test the content of it and the product respectively. By the HPLC experiment, we confirmed the TDC enzyme really works in our E.coli BL21 (DE3) system.

References

[1] Park S, Kang K, Lee S W, et al. Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli[J]. Applied Microbiology & Biotechnology, 2011, 89(5):1387-94

[2] Back K, Yin S, Chappell J (1994) Expression of a plant sesquiterpene cyclase gene in Escherichia coli. Arch Biochem Biophys 315:527–532

[3] Cabrita LD, Dai W, Bottomley SP (2005) A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. BMC Biotechnol 6:12

[4] Semba H, Ichige E, Imanaka T, Atomi H, Aoyagi H (2008) Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli in low-temperature culture. Appl Microbiol Biotechnol 79:563–569

[5] Steczko J, Donoho GA, Dixon JE, Sugimoto T, Axelrod B (1991) Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli. Protein Expr Purif 2:221–227