Overview
When carrying out our work, we did not just set our sights on the project itself, we have always been thinking about how to contribute to the future iGEM teams in the whole process and how to design a feasible solution for the world's common problem. Through unremitting efforts, we made some remarkable contributions in part registry and modeling work, which can provide references and ideas for the work of future iGEMers. Our products also have the potential to be developed into methods to achieve constipation prevention and relief in a biotherapeutic way, providing a template to address other issues.
Part collection
New part we have designed:
We have built several BioBricks, which cover all aspects throughout our projects, constituting our sensory module, metabolic module, and safety module.
Our basic parts
We designed six basic parts in total this year, integral to our cycle. You can click on the part to see details.
| Name | Type | Description |
|---|---|---|
| BBa K4656000 | Coding | pchA |
| BBa K4656001 | Coding | TDC |
| BBa K4656002 | Coding | TPH |
| BBa K4656003 | Coding | Lrp |
| BBa K4656004 | Promoter | Plee1 |
| BBa K4656005 | Promoter | PpchA |
Our composite parts
We designed seven composite parts in total this year, which are the backbone of our circuits. You can click on the part to see details.
| Name | Type | Description |
|---|---|---|
| BBa K4656007 | Composite | Plam-TPH-TDC |
| BBa K4656008 | Composite | pchA-PpchA-PLEE1-Cl |
| BBa K4656009 | Composite | Plac-tetR-Ptet-lacI-MazF |
| BBa K4656010 | Composite | PRM-Cl857-PR-phIF-MazE-PphIF-MazF |
| BBa K4656011 | Composite | pRha-tetR-tetO-mazF |
| BBa K4656012 | Composite | pT7-mleR-p_mleS-mazF |
| BBa K4656013 | Composite | pRha-mazE |
Part contribution
1.What our parts can do ?
The sensory module: Composed of pchA and Cl protein, can sense the concentration of butyrate and express the corresponding amount of Cl protein[1, 2].
The Cl protein has an inhibitory effect and can inhibit the metabolic module in a healthy human body[2].
The metabolic module: Composed of TPH and TDC, and was placed downstream of the plam promoter.
TPH and TDC are two enzymes that catalyze the production of 5-HT [3], which can bind to 5-HT receptors in the intestinal wall to promote intestinal motility and secretion[4], thereby improving constipation.
The safety module: Two lines consisting of drug control and temperature control.
Part1 drug control: We utilized bistable switch to achieve drug control when the engineered bacteria are in human body. If there is low dose of tetracycline or its analog atc in the environment, the plac promoter and its downstream tetR will be inhibited and a positive loop is formed to express MazF, thereby inducing bacterial death[5].
Part2 temperature control: Using toxin-antitoxin system, the engineered bacteria have different survival states in different scenarios.
In transportation scenario, the temperature is below 30°C with rhamnose addition, allowing for simultaneous expression of mazE and mazF, preventing bacterial death.
In in-vivo scenario, the temperature is 37°C, only mazE can be expressed, so the bacteria can survive.
In excretion scenario, the temperature is below 30°C and rhamnose is diluted. Only mazF can be expressed. Therefore the bacteria die. The backbone of our circuit is originated from Wang, et al[6].
2.part contribution
For contributions, we completed the experimental characterization of parts BBa_K4656007, BBa_K4656008, etc. and added data as well as documents to the corresponding BioBricks. All of these may be helpful to other teams.
In addition, our safety module is designed in a novel manner to control the survival or death of engineered bacteria by exogenous administration of TET or rhamnose, both to avoid overpopulation and to kill the engineered bacteria quickly when it is not wanted, in a controllable, human-initiated manner. Its combination with a temperature-controlled suicide system allows the engineered bacteria to die immediately after being expelled from the body without polluting the environment. Thus, we have provided the iGEM community with new parts along with new ideas for safe genetic routes.
Moreover, we added information to an existing part created by iGEM22_McMaster_Canada, clearly documenting our experiment design and data on the Part's Main Page on the Registry. Here shows the link BBa_K4442001.
Model
We introduced two new ideas in the modeling process. First, in the past, the choice of chassis for genetically engineered bacteria has often been based on experience or limited by clinical trials, which has led to the selection of chassis with sometimes disastrous consequences. We provide here an analytical approach based on Mendelian randomization that explores both the effect of the chassis on the disease and the effect of the disease on the chassis, which allows us to test whether our chassis is safe and feasible in an efficient way. Second, for E. coli growth and product production, in the past, people would use the method of smear plate or western blot for data collection, however, such experimental data collection has the disadvantages of being time-consuming, laborious, and less data. Therefore, to overcome these problems, we chose an enzyme labeler for automated data collection, which makes the fitting of our model more accurate.
Jump to Model page.
Product
Constipation is a common problem in modern life, with a prevalence rate of about 6%, meaning that 1 in 16 individuals suffer from constipation. Currently, there are five main treatment options for constipation, namely general therapy, medication, surgery, Chinese medicine and biofeedback therapy. However, these therapies are often time-comsuming and life-affecting, with unsatisfactory outcomes, and require self-motivation to take medication. Therefore, our team has decided to use synthetic biology to design a preventive, monitoring and therapeutic engineered bacteria using E. coli EcN as the chassis to help improve functional constipation. We plan to modify and make it carried by yogurt so that patients can implicitly interfere with constipation in their daily diet.
We designed sensory and metabolic modules to enable the engineered bacteria to express 5-HT based on the level of butyrate in the intestine to promote intestinal motility and secretion, thereby treating intestinal problems. We also added the safety module for urgent in vivo containment and environment protection. Accordingly, we performed wet experiments to validate for each module, and also built a model to fit the wet experiment results to provide reference for subsequent experiments.
The ideal process is as follows:
1.In the absence of constipation, butyrate concentration is normal in the intestine, and the sensory module continuously activates the expression of downstream Cl proteins, which inhibit downstream metabolic modules.
2.When constipation occurs, the concentration of butyrate in the intestine is significantly lower, and the expression of Cl proteins downstream of the sensory module is reduced, leading to a lowered inhibitory effect on the downstream metabolic module, thus activating the expression of serotonin synthesis-associated enzymes TPH1 and TDC.
3.The large amount of TPH1 and TDC produced promotes the increase of serotonin concentration in the intestines and acts on the intestinal 5-HT receptor 4, which promotes intestinal motility and secretion, so that the constipation symptoms can be relieved.
4.Use bistable switch to achieve drug control: when the engineered bacteria are in human body, if there is low dose of tetracycline and its analog atc in the environment, the plac promoter and its downstream tetR will be inhibited and a positive loop is formed to express toxin protein MazF and induce bacterial death.
5.Using toxin antitoxin system, the engineered bacteria have different survival states in different scenarios as aforementioned.
- Nakanishi N, Tashiro K, Kuhara S, Hayashi T, Sugimoto N, Tobe T. Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli. Microbiology (Reading). 2009;155(Pt 2):521-30. Epub 2009/02/10. doi: 10.1099/mic.0.023499-0. PubMed PMID: 19202100.
- Wang B, Kitney RI, Joly N, Buck M. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat Commun. 2011;2:508. Epub 2011/10/20. doi: 10.1038/ncomms1516. PubMed PMID: 22009040; PubMed Central PMCID: PMCPMC3207208.
- Park S, Kang K, Lee SW, Ahn MJ, Bae JM, Back K. Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli. Appl Microbiol Biotechnol. 2011;89(5):1387-94. Epub 2010/11/17. doi: 10.1007/s00253-010-2994-4. PubMed PMID: 21080162.
- Li B, Li M, Luo Y, Li R, Li W, Liu Z. Engineered 5-HT producing gut probiotic improves gastrointestinal motility and behavior disorder. Front Cell Infect Microbiol. 2022;12:1013952. Epub 2022/11/08. doi: 10.3389/fcimb.2022.1013952. PubMed PMID: 36339343; PubMed Central PMCID: PMCPMC9630942.
- Gardner TS, Cantor CR, Collins JJ. Construction of a genetic toggle switch in Escherichia coli. Nature. 2000;403(6767):339-42. Epub 2000/02/05. doi: 10.1038/35002131. PubMed PMID: 10659857.
- Wang X, Han JN, Zhang X, Ma YY, Lin Y, Wang H, et al. Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun. 2021;12(1):1411. Epub 2021/03/05. doi: 10.1038/s41467-021-21654-x. PubMed PMID: 33658500; PubMed Central PMCID: PMCPMC7930084.