iGEM 2021 — Team Shanghai_city

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# Team:Shanghai city/Notebook

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* [Team](https://2021.igem.org/Team%3AShanghai_city/Team)

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  + [Attributions](https://2021.igem.org/Team%3AShanghai_city/Attributions)
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* [Modeling](https://2021.igem.org/Team%3AShanghai_city/Model)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_city/Entrepreneurship)
* [Implementation](https://2021.igem.org/Team%3AShanghai_city/Implementation)
* [Human Practice](https://2021.igem.org/Team%3AShanghai_city/Human_Practices)

  + [Integrated Human
    Practice](https://2021.igem.org/Team%3AShanghai_city/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AShanghai_city/Communication)
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* [Parts](https://2021.igem.org/Team%3AShanghai_city/Parts)

  + [Parts
    Collection](https://2021.igem.org/Team%3AShanghai_city/Parts)
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* [Project](https://2021.igem.org/Team%3AShanghai_city/Description)

  + [Description](https://2021.igem.org/Team%3AShanghai_city/Description)
  + [Experiments](https://2021.igem.org/Team%3AShanghai_city/Experiments)
  + [Results](https://2021.igem.org/Team%3AShanghai_city/Results)
  + [Proof of Concept](https://2021.igem.org/Team%3AShanghai_city/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AShanghai_city/Notebook)
  + [Safety](https://2021.igem.org/Team%3AShanghai_city/Safety)
* [Home](https://2021.igem.org/Team%3AShanghai_city)

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# Notebook

Email Contact: *teamheartinker@gmail.com*

You can search “*Heartinker*” official Wechat account to know more about us!

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# Team:Shanghai city/Engineering

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# 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.

![](https://static.igem.org/mediawiki/2021/8/82/T--Shanghai_city--img_engineering_1.jpg)
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.

![](https://static.igem.org/mediawiki/2021/b/b4/T--Shanghai_city--img_engineering_2.jpg)

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).

# Learn

Genetically engineered bacteria produce TPH and TDC enzymes which are promising in treatment of
depression.

Email Contact: *teamheartinker@gmail.com*

You can search “*Heartinker*” official Wechat account to know more about us!

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# Team:Shanghai city/Experiments

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* [Team](https://2021.igem.org/Team%3AShanghai_city/Team)

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* [Modeling](https://2021.igem.org/Team%3AShanghai_city/Model)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_city/Entrepreneurship)
* [Implementation](https://2021.igem.org/Team%3AShanghai_city/Implementation)
* [Human Practice](https://2021.igem.org/Team%3AShanghai_city/Human_Practices)

  + [Integrated Human
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* [Parts](https://2021.igem.org/Team%3AShanghai_city/Parts)

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    Collection](https://2021.igem.org/Team%3AShanghai_city/Parts)
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  + [Contribution](https://2021.igem.org/Team%3AShanghai_city/Contribution)
* [Project](https://2021.igem.org/Team%3AShanghai_city/Description)

  + [Description](https://2021.igem.org/Team%3AShanghai_city/Description)
  + [Experiments](https://2021.igem.org/Team%3AShanghai_city/Experiments)
  + [Results](https://2021.igem.org/Team%3AShanghai_city/Results)
  + [Proof of Concept](https://2021.igem.org/Team%3AShanghai_city/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AShanghai_city/Notebook)
  + [Safety](https://2021.igem.org/Team%3AShanghai_city/Safety)
* [Home](https://2021.igem.org/Team%3AShanghai_city)

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# Experiments

Email Contact: *teamheartinker@gmail.com*

You can search “*Heartinker*” official Wechat account to know more about us!

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai city/Results

[![](https://static.igem.org/mediawiki/2021/4/4c/T--Shanghai_city--logo.png)](https://2021.igem.org/Team%3AShanghai_city)

* [Team](https://2021.igem.org/Team%3AShanghai_city/Team)

  + [Team Members](https://2021.igem.org/Team%3AShanghai_city/Team)
  + [Attributions](https://2021.igem.org/Team%3AShanghai_city/Attributions)
  + [Collaboration](https://2021.igem.org/Team%3AShanghai_city/Collaborations)
* [Modeling](https://2021.igem.org/Team%3AShanghai_city/Model)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_city/Entrepreneurship)
* [Implementation](https://2021.igem.org/Team%3AShanghai_city/Implementation)
* [Human Practice](https://2021.igem.org/Team%3AShanghai_city/Human_Practices)

  + [Integrated Human
    Practice](https://2021.igem.org/Team%3AShanghai_city/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AShanghai_city/Communication)
  + [Fundraising](https://2021.igem.org/Team%3AShanghai_city/Fundraising)
* [Parts](https://2021.igem.org/Team%3AShanghai_city/Parts)

  + [Parts
    Collection](https://2021.igem.org/Team%3AShanghai_city/Parts)
  + [Engineering](https://2021.igem.org/Team%3AShanghai_city/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AShanghai_city/Contribution)
* [Project](https://2021.igem.org/Team%3AShanghai_city/Description)

  + [Description](https://2021.igem.org/Team%3AShanghai_city/Description)
  + [Experiments](https://2021.igem.org/Team%3AShanghai_city/Experiments)
  + [Results](https://2021.igem.org/Team%3AShanghai_city/Results)
  + [Proof of Concept](https://2021.igem.org/Team%3AShanghai_city/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AShanghai_city/Notebook)
  + [Safety](https://2021.igem.org/Team%3AShanghai_city/Safety)
* [Home](https://2021.igem.org/Team%3AShanghai_city)

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# Results

# Recombinant E. coli

## Construction of plasmid pTRC99K-5HT

1. Preparation of plasmid vector pTRC99K

![](https://static.igem.org/mediawiki/2021/2/2e/T--Shanghai_city--img_results_2.jpg)
Figure 1. DNA concentration comparing with the pure water

The DNA concentrations of four tubes plasmid pTRC99K are 325.5ng/μl, 386.6ng/μl, 234.3ng/μl,
225.1ng/μl, respectively.

2. Acquisition of linearized vectors pTRC99K and inserts TDC-TPH

![](https://static.igem.org/mediawiki/2021/1/16/T--Shanghai_city--img_results_3.jpg)

* 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

3. Gel electrophoresis of recombinant plasmid pTRC99K- TDC-TPH

![](https://static.igem.org/mediawiki/2021/b/bb/T--Shanghai_city--img_results_4.jpg)

* 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.

4. Rcombinant plasmid pTRC99K- TDC-TPH sequencing analysis

Global sequence alignment of sequencing:

![](https://static.igem.org/mediawiki/2021/d/d5/T--Shanghai_city--img_results_5.jpg)

The details of TPH sequence alignment:

![](https://static.igem.org/mediawiki/2021/6/60/T--Shanghai_city--img_results_6.jpg)

The details of TDC sequence alignment:

![](https://static.igem.org/mediawiki/2021/c/c7/T--Shanghai_city--img_results_7.jpg)

The sequencing results show that both TPH and TDC are inserted into pTRC99K successfully.

5. Expression of TPH and TDC proteins

![](https://static.igem.org/mediawiki/2021/a/ab/T--Shanghai_city--img_results_8.jpg)
Figure 2: SDS-PAGE analysis for TPH and TDC proteins.

1. Lane 1: Culture supernatant with induction (40 μL)；
2. Lane 2: Culture supernatant without induction (40 μL)；
3. Lane 3: Without samples
4. Lane 4: Supernatant of cell lysate with induction (40 μL)；
5. Lane 5: Supernatant of cell lysate without induction (40 μL)；
6. Lane 6: Pellet of cell lysate with induction (40 μL)；
7. Lane 7: Pellet of cell lysate without induction (40 μL)；
8. Lane 8: Supernatant of cell lysate with induction (20 μL)；
9. 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 expressed successfully.

# Rin 14b cell screening platform

## Patch Clamp Results

![](https://static.igem.org/mediawiki/2021/d/db/T--Shanghai_city--img_results_9.jpg)
![](https://static.igem.org/mediawiki/2021/1/13/T--Shanghai_city--img_results_10.jpg)

After administration, the membrane potential became stable and the release frequency of AP increased,
suggesting that this compound may have the ability to stably activate RIN14B cells.

![](https://static.igem.org/mediawiki/2021/b/b8/T--Shanghai_city--img_results_11.jpg)

After administration, the membrane potential decreased, the release frequency of AP increased, and
the amplitude increased significantly, and the effect could not be eluted, which may indicate that
this compound has a strong activation ability for RIN14B cells.

![](https://static.igem.org/mediawiki/2021/8/82/T--Shanghai_city--img_results_12.jpg)

The membrane potential and AP release frequency increased after administration, suggesting that the
compound may have the ability to activate RIN14B cells.

Email Contact: *teamheartinker@gmail.com*

You can search “*Heartinker*” official Wechat account to know more about us!