Part:BBa_K3999000
TPH
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1606
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Profile
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
Experiment Data
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.
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.
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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