Coding

Part:BBa_K3999000

Designed by: Ke ZHU   Group: iGEM21_Shanghai_city   (2021-10-11)
Revision as of 10:54, 21 October 2021 by TheaW (Talk | contribs) (→‎Properties: BBa_K3999000 is the coding sequence of tryptophan hydroxylase (TPH), which catalyzes the L-tryptophan to the 5-hydroxy-L-tryptophan)


TPH


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1606
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: TPH

Base Pairs:1641bp

Origin: Synthetic

Properties: the coding sequence of tryptophan hydroxylase (TPH). =

Usage and biology

TPH catalyzes the L-tryptophan to the 5-hydroxy-L-tryptophan

Figure1. The protein structure of tryptophan 5-monooxygenase monomer, Human..

Experiment Data

Acquisition of linearized vectors pTRC99K and inserts TDC-TPH

Figure2. 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

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

Figure3. The details of TPH 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.

Figure4.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.

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