LZU-HS-Pro-A

Overview

Synthetic biology generally refers to the design and construction of new biological elements, devices, and systems. It is a kind of redesigning existing biological systems for specific purposes. Research in synthetic biology follows the DBTL cycle (design, build, test, learn), which is an iterative process to develop an innovative biological system for the ultimate purpose of application.

iGEM Engineering Cycle

The probiotics designed by our team to achieve the purpose of treating depression is the core goal of our project. The introduction of this system follows the complete cycle of "design - build - test - learn". First, we designed Crisper Cas9 to insert a gene that produces antidepressants into the probiotic Escherichia coli Nissle 1917, which function in the small intestine. Then we carried out relevant experiments based on the designed gene circuit, and successfully cultivated the strain. Subsequently, we carried out functional tests on the transgenic bacteria, and found that the successfully constructed engineering strain only produced substances in the cell and could not be discharged in large quantities in vitro, which made it difficult to achieve the treatment of depression. After consulting relevant reference and communicating with the other teams, we were inspired to build a complete tea lysis system by improving the design method of bacterial engineering. Our expression system still makes it possible to use it as a new depression drug and ultimately achieve our goal.

Project

Design:

Our mental probiotics mainly need to insert two types of genes, one is the human tryptophan hydroxylase I (TPH1) gene, the other is the glutamic acid decarboxylase gene, both of which produce 5-HTP (a precursor of serotonin), the other is γ-aminobutyric acid.

Gene Circuit of our Project

Build:

IGEM Engineering Cycle—BUILD

We plan to activate the bacteria first, and then extract the plasmids, which will be amplified by PCR after extraction, and then recycle the plasmids successfully digested by gel through double digestion and nucleic acid electrophoresis. The target cells were activated to the competent state, and the link reaction was carried out with the target gene. Finally, the positive clone was obtained, and the engineered vector was transformed into the expression host E. coli BL21.

Gel Electrophoresis

Test:

iGEM Engineering Cycle—TEST

In our project, after the construction of the transgenic strain, we need to verify whether the strain can successfully express the required substances, 5-HTP and γ-aminobutyric acid.

We first observed the fluorescence transfection of the strain under the fluorescence microscope, and preliminarily confirmed that the engineered bacteria could express 5-HTP and γ-aminobutyric acid.

Fluorescent Transfection

To further accurately detect the expression amount of 5-HTP and GABA, we use ELISA (immunoassay (IA)) technique. ELISA technique is performed by solid phase of antigen or antibody and enzymatic labeling of antigen or antibody, and after adding the substrate of enzyme reaction, the substrate is catalyzed by enzyme to become product with specific color, and the amount of product is observed for qualitative or quantitative analysis. Therefore, we choose this technique for further detection of expression.

Successful expression of γ-aminobutyric acid in Nissle1917

The amount of GABA was given in mg/L units. In order to verify the expression, the strains after lysis and those before lysis were used for comparison.

Successful expression of 5-HTP in Nissle1917

The amount of 5-HTP is given in mg/L units and to verify expression, both post-cleavage strains and pre-cleavage strains were used for comparison.

The experimental results demonstrated that the engineered strain we constructed could effectively produce GABA and 5-HTP, but because most of the two substances were not secreted outside the bacterium, resulting in far more fragmented organisms than the non-fragmented group. We hypothesized that the synthesis path of 5-HTP was too long, or the culture environment provided was not optimal. In this regard, we designed experiments to test and verify the optimal pH and temperature of the engineered strain, and to test whether the modified strain can survive in the human body.

We cultured engineered strains in LB liquid medium and measured GABA and 5-HTP content after 48 h of incubation at 37 ° C, N=3.

Expression of GABA and 5-HTP at 37 ° C and N=3

The experimental results showed that the optimal survival temperature of the successfully constructed strain was 37 degrees Celsius, and the optimal pH value was about 7, which was roughly the same as the temperature and pH value in the intestine, and the strain could survive and function in the human body.

Although we verified the two most important factors for a strain's survival in the gut, there were also effects such as the remaining flora in the gut, the presence of digestive fluids such as gastric and intestinal fluids, so we roughly simulated the gut environment: The main simulation conditions were HP =7, the temperature was 37 ° C, bile salts and pancreatic fluid were added, and the culture was conducted under hypoxia (hypoxia bag used) and the GABA expression was detected by ELISA (because the amount of 5-HTP was too small, the expression of 5-HTP was not measured in this experimental verification).

Successful expression of GABA in the simulated intestinal environment

It was verified that both control and mock groups could survive in the simulated intestinal environment, and the yield of the mock group was significantly increased compared with the control group.

Learn:

iGEM Engineering Cycle--LEARN

From the above test results, it can be found that GABA and 5-HTP are both in the cell. Although GABA and 5-HTP can be transported by themselves across the membrane, the secretion rate of GABA and 5-HTP by the strain itself is too slow and the amount is too small. However, the absorption of GABA and 5-HTP in human body requires the release of a large amount of GABA and 5-HTP by thallus. We have provided solutions to this problem through cooperation and communication with other teams and reference review.

Design:

iGEM Engineering Cycle--DESIGN

Solution: Tea polyphenols and phenolic acids in tea will be metabolized to protocatechuic acid (PCA). We used protocatechuic acid (PCA) -specific promoters to ligate lysis genes.

We selected the SRRz lysis gene to create our lysis module. The SRRz gene was derived from bacteriophage. It is composed of S, R and Rz.

After consumers take our probiotics, the probiotics will produce GABA and 5-HTP. When we drink rea, the protocatechuic acid in tea will start the lysis system, and GABA and 5-HTP will be released into the intestine after bacterial lysis.

Build:

iGEM Engineering Cycle—DESIGN

Due to the limited time, we do the test on the previously cultivated Nissle 1917 engineering bacteria. The chassis microorganism used was BL21, and this engineering strain only had lysis module.

Protocatechuic acid (PCA) promoter 1

Protocatechuic acid (PCA) promoter 2

Test:

iGEM Engineering Cycle—TEST

We cultured the engineered strain in the LB medium and put different concentrations of pro-catecholic acid (PAC) in it while performing replicate experiments.

Expression effect of pro-catecholic acid lysis system in bacteria, higher OD600 translucency indicates higher lysis

The experimental results showed that when the concentration of protocatechuic acid (PCA) in the intestine can be controlled around 10-7 mol, it can ensure the partial lysis and partial survival of the bacterium. The partial lysis of the bacterium can release part of GABA and 5-HTP, while the surviving part of the bacterium can continue to reproduce.

The results of this experiment proved the feasibility of our constructed protocatechuic acid promoter in the strain; although it was not operated on the basis of the original engineered strain, the experiment can still prove the feasibility of the theory.

Learn:

iGEM Engineering Cycle-- LEARN

Base on two cycles, in order to increase biosecurity, we designed a suicide system on the basis of the original lysis system.

The lactose operon is a macromolecular operon that exists both inside and outside the body, so it is difficult to regulate specific genes. We found a relatively perfect two killing switches in the paper "Structure and function of bacterial kid-kis and related toxin-antitoxin systems". Chemical response switches and input chemical and temperature response switches, while improving the stability of the switch. The chemical response switch initiates the death switch in response to the chemical inducer anhydrous tetracycline (aTc), and the termination switch initiates additional death due to a sudden drop in temperature caused by host excretion. For the chemical start switch, it is based on the design of CRIPER, gRNA is expressed through the ATC-inducible promoter Ptet, and TetR is expressed after gRNA, which needs the promoter Ptet. In the presence of aTc, TetR cannot bind to the target promoter, and the cell will initiate the Pet promoter to express CAS9 and gRNA. The selection of optimized 2-grNA for expression can effectively reduce the accidental killing and improve the stability.

TlpA is a transcriptional regulator that aids transcription of PtlpA at low temperatures (experimental setting: 33 degrees Celsius) while allowing transcription to occur at high temperatures (experimental setting: 37 degrees Celsius).

The result is that when the modified Nissle 1917 escapes and is excreted by the patient, it activates the kill switch that causes the bacteria to commit suicide. If it is necessary to stop E. coli from functioning, we can arrange the patient to take oral anhydrous tetracycline antibiotics on the basis of the previous experiment, and perform collective elimination of probiotics in the gut.

Conclusion

Through a complete design-build-test-learning process, our team independently designed gene circuits to construct engineered strains producing 5-HTP and GABA, completed the construction and conducted functional tests. After discovering the problem, we actively learned to design a feasible lysis system and tested it. So that's the part of the engineering success of our team.

Reference

[1] Yin, Jianli, 等. 《A green tea–triggered genetic control system for treating diabetes in mice and monkeys》. Science Translational Medicine, 卷 11, 期 515, 2019年10月, 页 eaav8826. science.org (Atypon), https://doi.org/10.1126/scitranslmed.aav882

[2]. Kamphuis, M. B., Monti, M. C., van den Heuvel, R. H., López-Villarejo, J., Díaz-Orejas, R., & Boelens, R. (2007). Structure and function of bacterial kid-kis and related toxin-antitoxin systems. Protein and peptide letters, 14(2), 113–124. https://doi.org/10.2174/092986607779816096