Difference between revisions of "Part:BBa K3999004"

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pTrc99k-TPH-TDC

pTrc99k-TPH-TDC

Profile

BBa_K3999004

Name: pTrc99k-TPH-TDC

Base Pairs:6839bp

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.

Construct design

Figure1. Schematic map of pTrc99k-TPH-TDC.

After researching, we found the humans use two enzymes, tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC), to convert Tryptophan(Trp), which is an essential amino acid for human’s body, to 5-HT. Moreover, TPH is the rate-limiting enzyme in the process, which means the amount of TPH can directly affect the production speed of 5-HT (Liu 2021). Thus, We hope to modify E. coli by inserting TPH and TDC genes, so that it can become the exogenous synthesis pathway of 5-HT, so as to increase the amount of 5-HT.

BBa_K3999000

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

Figure2. The protein structure of tryptophan 5-monooxygenase monomer, Human..

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.

Figure4. Plasmid map of pTRC99K.

Experiment Data

Figure5.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.

Rcombinant plasmid pTRC99K- TDC-TPH sequencing analysis

Figure6.Global sequence alignment of sequencing:.

Sequence and Features