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).

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 linked TDC with the vector pET15(+), which has N-terminal His tag that makes 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( DE3):

When the plasmid was constructed successfully, we must detect whether the enzyme we obtained can express in E.coli system. Therefore, we transferred the pET15-TDC plasmid into E.coli BL21 (DE3) strain [3] for expression. What’s more, in order to find out where the protein exactly existed, 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, in order 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 that the TDC enzyme really worked 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

Characterization by 2021iGEM_Shanghai_city

Improvement of an existing part

Compared to the old part BBa_K2276001, which consisting of T7 promotor, 6xHis tag, thrombin site, and the coding sequence of tryptophan decarboxylase (TDC). We found the humans use another enzymes, tryptophan hydroxylase (TPH), to convert Tryptophan(Trp), which is an essential amino acid for human’s body, to 5-HT. So we design a new part BBa_K3999004. Moreover, TPH is the rate-limiting enzyme in the process, which means the amount of TPH can directly affect the production speed of 5-HT (Liu 2021). Thus, We hope to modify E. coli by inserting TPH and TDC genes, so that it can become the exogenous synthesis pathway of 5-HT, so as to increase the amount of 5-HT.

Figure 13.EC cell releases 5-HT.

We were inspired by a fascinating property of 5-HT that less than one in a million CNS neurons produce serotonin, and 95% of serotonin was released into the gut by intestinal enterochromaffin cells (Berger 2018). Hence, we hope to stimulate the EC cell to make it release more 5-HT in the blood and increase the chance to treat the depression via a gut-brain circuit. As Dr. Nozawa et al. (2009) reported in their journal, TRPA1 is an excitatory ion channel that can be stimulated by many natural compounds, like spices and herbal medicines, and these TRPA1 agonists cause Ca2+ influx and 5-HT release in EC cells. Luckily, this ion channel is also highly expressed in gastrointestinal tissues in humans, mice, and rats. In other words, if we can stimulate the TRPA1 agonists on the EC cells, we can make it release more 5-HT. However, it’s impossible to achieve a completely pure EC cell culture to test. Then we turn our attention to another cell with a similar function as the EC cell, the Rin 14b cell. Dr. Nozawa and his colleagues found the gene expression markers of EC cells such as TPH1, chromogranin A, VMAT1, and synaptophysin are also highly expressed in Rin 14b cells. Moreover, the agonists, like Ca2+ and ionomycin, which can trigger the release of 5-HT from EC cells, can also activate Rin 14b cells, indicating that RIN14B cells share functional similarities with EC cells and can be used as a model to test the potential compounds. In this part of our project, we will use the patch-clamp techniques to find compounds that can stimulate the Rin 14b cells, which can increase the attitude and frequency of its action potential and mark it as a possible direction that pharmacologists can keep researching on.

Figure 14.The blast results about the TDC's amino acid sequence of our new part BBa_K3999004 and the old part BBa_K2276001.

BBa_K3999004

Properties

Overexpression of tryptophan hydroxylase and 5-hydroxytryptophan decarboxylase

Usage and Biology

According to the World Health Organization(2020), depression became a major contributor to disease burden, with more than 264 million people suffering from its pain. People are constantly under stress from various aspects of life: career, socialization, study, etc. Unfortunately, in December 2019, an outbreak of CoronaVirus Disease 2019 (Covid-19) ravaged the world; nearly four billion people died during this pandemic. (Ritchie 2021). Under such an intense environment, many friends and relatives of ours fall victim to depression. Yunhe Wang, et al. (2021) used a national online survey demonstrating that people who experienced quarantine have a higher risk of being influenced by depression, especially those who have a history of mental illness and are infected by Covid-19. These heartbreaking numbers bring students worldwide with ambition in biology and psychology to Shanghai and form this team. We, the Heartinker team, are deeply concerned about the situation of depression patients and hope to use the advanced technology of synthetic biology to discover a new antidepressive target and find a more effective and harmless antidepressive product to solve the problems of current antidepressants. Serotonin is widely found in the natural tissues of animals, especially in the cerebral cortex and synapses in high levels, and also in plants and fungi in a small amount. In the pharmaceutical field, serotonin, as a drug, can participate in a variety of physiological functions of the organism, including emotion regulation, behavior management, sleep cycle maintenance, scavenging harmful free radicals and so on. The Monoamine hypothesis is a widely accepted hypothesis for the cause of depression at present. This theory holds that depression is caused by the decrease in the concentration or function of monoamine neurotransmitters (such as 5-hydroxytryptamine) in the synaptic space of the central nervous system. The decrease of the neurotransmitter function of 5-hydroxytryptamine(Serotonin or 5-HT) not only leads to the occurrence of depression and anxiety, but also interferes with the normal function of other neural circuits. Even though the deficiency of 5-HT can lead to depression is still a hypothesis, after decades of research, the role of 5-HT in depression has been refined, and more scientific evidence suggests the importance of 5-HT in treating depression (Albert 2012). Therefore, we decided to choose 5-HT as a direction to develop our antidepressive product in two pathways.

Construct design

Figure1. Schematic map of pTrc99k-TPH-TDC.

After researching, we found the humans use two enzymes, tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC), to convert Tryptophan(Trp), which is an essential amino acid for human’s body, to 5-HT. Moreover, TPH is the rate-limiting enzyme in the process, which means the amount of TPH can directly affect the production speed of 5-HT (Liu 2021). Thus, We hope to modify E. coli by inserting TPH and TDC genes, so that it can become the exogenous synthesis pathway of 5-HT, so as to increase the amount of 5-HT.

BBa_K3999000

Name: TPH Base Pairs:1641bp Origin: Synthetic Properties: BBa_K3999000 is the coding sequence of tryptophan hydroxylase (TPH), which catalyzes the L-tryptophan to the 5-hydroxy-L-tryptophan

Figure2. The protein structure of tryptophan 5-monooxygenase monomer, Human..

BBa_K3999001

Properties

BBa_K3999001 is the coding sequence of the tryptophan decarboxylase (TDC), a kind of aromatic L-amino acid decarboxylase (AADC or AAAD). The AADC also known as DOPA decarboxylase (DDC), tryptophan decarboxylase, and 5-hydroxytryptophan decarboxylase, is a lyase enzyme (EC 4.1.1.28). AADC catalyzes several different decarboxylation reactions.

In our project, TDC is used to catalyze 5-Hydroxytryptophan (5-HTP) to serotonin (5-hydroxytryptamine, 5-HT). In normal dopamine and serotonin (5-HT) neurotransmitter synthesis, AADC is not the rate-limiting step in either reaction. However, AADC becomes the rate-limiting step of dopamine synthesis in patients treated with L-DOPA (such as in Parkinson's disease), and the rate-limiting step of serotonin synthesis in people treated with 5-HTP (such as in mild depression or dysthymia). AADC is inhibited by carbidopa outside of the blood-brain barrier to inhibit the premature conversion of L-DOPA to dopamine in the treatment of Parkinson's.

Figure3. Ribbon diagram of a DOPA decarboxylase dimer..

BBa_K3999002

Properties

BBa_K3999002 is a plasmid backbone. This sequence contains multiple cloning sites (MCS), used for restriction digest assembly of our pTRC99K plasmids. It also contains the promoter, coding sequence, and terminator for streptomyces kanamyceticus resistance (KanR). It contains an origin of replication in E.coli for the plasmid, a LacI promoter, a LacI coding sequence for a lac repressor, a Lac operator, and a trp promoter within lacUV5.

Figure4. Plasmid map of pTRC99K.

Experiment Data

Figure5.Gel electrophoresis of recombinant plasmid pTRC99K- TDC-TPH.

Line 4: control plasmid, 6379bp

Gel electrophoresis of recombinant plasmid pTRC99K- TDC-TPH shows that the size of pTRC99K-TDC-TPH-2 is correct.

Rcombinant plasmid pTRC99K- TDC-TPH sequencing analysis

Figure6.Global sequence alignment of sequencing:.

Figure7.The details of TPH sequence alignment:.

Figure8.The details of TDC sequence alignment:.

The sequencing results show that both TPH and TDC are inserted into pTRC99K successfully.

In theory, the expression of inserted coding sequence must be induced by IPTG. However, experiment data shows that protein can express successfully regardless of whether there is IPTG induction.

Figure 9: SDS-PAGE analysis for TPH and TDC proteins..

Lane 9: Supernatant of cell lysate without induction (20 μL)；

We got two bands 43.53kDa (TPH) and 53.55kDa (TDC) in both supernatant of cell lysate and pellet of cell lysate. These results suggest that TPH and TDC proteins can be expressed successfully regardless of IPTG induction.

Proof of function

Patch Clamp Results

table 1：Summary of Compounds.

Figure 10.The map of results.

After administration, the membrane potential became stable and the release frequency of AP increased, suggesting that this compound may have the ability to stably activate RIN14B cells.

Figure 11.The map of results.

After administration, the membrane potential decreased, the release frequency of AP increased, and the amplitude increased significantly, and the effect could not be eluted, which may indicate that this compound has a strong activation ability for RIN14B cells.

Figure 12.The map of results.

The membrane potential and AP release frequency increased after administration, suggesting that the compound may have the ability to activate RIN14B cells.

(5)PJ;, H. C. J. D. R. S. C. (2017, May 4). How do antidepressants work? New perspectives for refining future treatment approaches. The lancet. Psychiatry. https://pubmed.ncbi.nlm.nih.gov/28153641/.

===(6)Shulman, K. I., Herrmann, N., & Walker, S. E. (2013, August 10). Current Place of Monoamine Oxidase Inhibitors in the Treatment of Depression. CNS Drugs. https://link.springer.com/article/10.1007/s40263-013-0097-3.

(9)[1] Côté F, Thévenot E, Fligny C, Fromes Y, Darmon M, Ripoche MA, et al. (November 2003). "Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function". Proceedings of the National Academy of Sciences of the United States of America. 100 (23): 13525–30. Bibcode:2003PNAS..10013525C. doi:10.1073/pnas.2233056100. PMC 263847. PMID 14597720.

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]