Contribution
==BBa_K3999000==
BBa_K3999000 is the coding sequence of tryptophan hydroxylase (TPH), which catalyzes the L-tryptophan to the 5-hydroxy-L-tryptophan. As the rate-limiting enzyme of 5-HT synthesis, TPH plays an important role in invertebrate neuroregulation. In nature, serotonin is widely present in mammalian tissues, especially in the cerebral cortex and nerve synapses, and has a small amount in plants and fungi. In the pharmaceutical field, serotonin, as a drug, can participate in a variety of physiological functions of the organism, including emotion regulation, behaviour management, sleep cycle maintenance, and removal of harmful free radicals.
Figure1. The protein structure of tryptophan 5-monooxygenase monomer, Human.
==BBa_K3999001==
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.
Figure2. Ribbon diagram of a DOPA decarboxylase dimer.
==BBa_K3999002==
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.
Figure3. Plasmid map of pTRC99K
===Experiment Data===
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.
==BBa_K3999003==
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.
Figure1. Human serotonin (5-HT) biosynthesis pathway.
==Reference==
[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.
==BBa_K3999004==
In order to synthesize serotonin(5-hydroxytryptamine, 5-HT) in our recombinant E.coli strains, we design this plasmid BBa_K3999004. The coding sequence of tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC) is inserted at the multiple cloning site on pTRC99K. The pTRC99K-vector is used for recombinant protein TPH and TDC production in recombinant E.coli. The expression of TPH and TDC be induced by IPTG. Then the formation of active enzymes catalyzes L-tryptophan to 5-HT.
Figure5. The plasmid map of pTRC99K-TPH-TDC.