Engineering

Background

According to the statistics of World Health Organization satitcs (2020), depression became a major contributor to burden of disease, with more than 264 million people suffering from it. People are under stress due to various aspects of life, for example career, socialization, study, in particular of outbreak of Coronavirus Disease 2019 (COVID-19). People are deeply concerned about the situation of depression patients and hope to use the biotechnology to discover a new antidepressive approaches to solve the consequence of depression.

It is a widely accepted that the cause of depression is the decrease in the concentration or function of monoamine neurotransmitters (such as 5-hydroxytryptamine, 5-HT) in the synaptic space of the central nervous system. The decrease of the 5-HT not only leads to the occurrence of depression and anxiety, but also interferes with the normal function of other neural circuits. Scientific evidence also suggested the importance of 5-HT in treating depression. Therefore, we decided to choose 5-HT as target to develop antidepressive products.

Design

There are two enzymes, tryptophan hydroxylase (TPH) and tryptophan decarboxylase (TDC), to convert essential amino acid Tryptophan (Trp) to 5-HT. TPH is the rate-limiting enzyme in the process, which means the amount of TPH can directly affect the production speed of 5-HT. Thus, genetically engineered Escherichia coli which contain exogenous TPH and TDC genes was constructed so that it can become the exogenous synthesis pathway of 5-HT, so as to increase the amount of 5-HT.

Build

The recombinant plasmids were constructed to produce recombinant TPH and TDC in cells (Figure 1). TPH and TDC genes were inserted in pTrc99k which are controlled by P1 promoter and rrB terminators. The sequencing results show that both TPH and TDC are inserted into pTRC99K successfully.

Figure 1. Schematic map of plasmids.

Test

SDS-PAGE shows TPH and TDC expression in E. coli (Figure 2). 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 expressed successfully.

Figure 2. SDS-PAGE analysis of the expression of TPH and TDC. 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).

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