Coding

Part:BBa_K3999001

Designed by: Ke ZHU   Group: iGEM21_Shanghai_city   (2021-10-11)


TDC

TDC

Profile

Name: TDC

Base Pairs:1482bp

Origin: Synthetic

Properties:the coding sequence of the tryptophan decarboxylase (TDC).

Usage and Biology

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.

Figure1. Ribbon diagram of a DOPA decarboxylase dimer.

Experiment Data

Figure2.Gel electrophoresis of recombinant plasmid pTRC99K- TDC-TPH.

Line 1: pTRC99K-vector, control plasmid, 4167bp

Line 2: recombinant plasmid pTRC99K-TDC-TPH-1, incorrect

Line 3: recombinant plasmid pTRC99K-TDC-TPH-2, 7077bp, correct

Line 4: control plasmid, 6379bp

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

Figure3.The details of TDC sequence alignment:.

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 4: SDS-PAGE analysis for TPH and TDC proteins.

Lane 1: Culture supernatant with induction (40 μL)；

Lane 2: Culture supernatant without induction (40 μL)；

Lane 3: Without samples

Lane 4: Supernatant of cell lysate with induction (40 μL)；

Lane 5: Supernatant of cell lysate without induction (40 μL)；

Lane 6: Pellet of cell lysate with induction (40 μL)；

Lane 7: Pellet of cell lysate without induction (40 μL)；

Lane 8: Supernatant of cell lysate with induction (20 μL)；

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.

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 319
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 340
    Illegal XhoI site found at 850
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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