Coding

Part:BBa_K3999003

Designed by: Ke ZHU   Group: iGEM21_Shanghai_city   (2021-10-12)
Revision as of 08:57, 18 October 2021 by Zhuke18 (Talk | contribs)


TPH-TDC

TPH-TDC

Profile

Name: TPH-TDC

Base Pairs:3192bp

Origin: Synthetic

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.

Figure1.Human serotonin(5-HT)biosynthesis pathway.

Construct and Design

Figure 2. TDC and TPH protein expression box.

BBa_K3999003

Name: TPH-TDC

Base Pairs:3192bp

Origin: Synthetic

Properties

Overexpression of tryptophan hydroxylase and 5-hydroxytryptophan decarboxylase

In animals including humans, serotonin(5-hydroxytryptamine, 5-HT) is synthesized from the amino acid L-tryptophan by a short metabolic pathway consisting of two enzymes, tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase (DDC), and the coenzyme pyridoxal phosphate. The TPH-mediated reaction is the rate-limiting step in the pathway.[1]

In order to synthesize 5-HT in our recombinant E.coli strains, we design this composite part BBa_K3999003, which contains the coding sequence of tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC). The TPH gene and the TDC gene can be expressed in recombinant E. coli. Then the formation of two active enzymes TPH and TDC can catalyze L-tryptophan to 5-HT.

BBa_K3999001

Name: TDC

Base Pairs:1482bp

Origin: Synthetic

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

Name: pTrc99k

Base Pairs:4167bp

Origin: Synthetic

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.

Experiment Data

Acquisition of linearized vectors pTRC99K and inserts TDC-TPH

Figure4. Acquisition of linearized vectors pTRC99K and inserts TDC-TPH.
Line 1: pTRC99K-vector, control plasmid
Line 2: linearized pTRC99K-vector, correct
Line 3: the PCR product of TDC, correct
Line 4: the PCR product of TPH, correct
Line 5: the over-lap PCR product of TDC-TPH, correct
Line 6: the over-lap PCR product of TDC-TPH, correct

Reference

(1)Gillman, P. K. (2009, January 29). BPS Publications. British Pharmacological Society | Journals. https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1038/sj.bjp.0707253#b88.

(2)J, N. D., S, A., J, N., J, S., A, B., & S, F. (1999, July 9). Mechanisms of action of selective serotonin reuptake inhibitors in the treatment of psychiatric disorders. European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology. https://pubmed.ncbi.nlm.nih.gov/10523062/.

(3)V, R. M., & Z, P. W. (1999, June 19). Metabolism of tricyclic antidepressants. Cellular and molecular neurobiology. https://pubmed.ncbi.nlm.nih.gov/10319193/.

(4)Gillman, P. K. (2009, January 29). BPS Publications. British Pharmacological Society | Journals. https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1038/sj.bjp.0707253.

(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.

(7)Du, Y., Gao, X.-R., Peng, L., & Ge, J.-F. (2020). Crosstalk between the microbiota-gut-brain axis and depression. Heliyon, 6(6). https://doi.org/10.1016/j.heliyon.2020.e04097

(8)Nozawa, K., Kawabata-Shoda, E., Doihara, H., Kojima, R., Okada, H., Mochizuki, S., Sano, Y., Inamura, K., Matsushime, H., Koizumi, T., Yokoyama, T., & Ito, H. (2009). TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells. Proceedings of the National Academy of Sciences, 106(9), 3408–3413. https://doi.org/10.1073/pnas.0805323106

(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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1960
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1606
    Illegal XhoI site found at 1981
    Illegal XhoI site found at 2491
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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