Overview
Synthetic biology is the utilization of engineering principles, using synthetic DNA, gene editing and bioengineering technology to design, build and improve biological systems to achieve the goal of application. Therefore, after using these engineering principles to design and build prototypes, it is necessary to go through a series of tests and repeated improvements to ensure that the final product meets the initial expectations and achieves the optimization of application. Our team applied this concept to the project, forming a complete engineering cycle :research, imagine, design, build, test and learn. In the research, we conducted literature review and expert consultation. In imagine, team members carried out brainstorming and put forward creative ideas. In the design, according to the objectives, scope, feasibility and resource requirements of the project, we used engineering principles to design gene fragments and verification methods. In the build, we sent the designed gene fragment to the company for synthesis, and the synthesized plasmid was transferred into the chassis bacteria. In the test, we analyzed the function of the engineering biological system we constructed through bacterial experiments. In the process of learning, we analyzed the experimental results to evaluate whether the engineering biological system we constructed has achieved the expected goal. Through the above several engineering cycles, we have made gut-sweeper into a safe and effective engineering E.coli, which provides a new idea for the treatment of constipation.
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Abstract
In the initial phase of the project during the first engineering cycle, we endeavored to identify characteristic substance changes in the body when it experiences constipation. Through investigation and research, we observed that an increase in methane gas and a decrease in butyrate concentration occur within the intestines when the body is constipated. Consequently, we initially designated methane and butyrate as sensing biomarkers for our engineered bacteria. However, later findings from interviews with relevant experts and literature research revealed that certain normal physiological phenomena could also lead to elevated methane levels, and the specificity of methane in relation to constipation was not strong. Furthermore, methane needs to be converted into methanol to be sensed by the engineered bacteria, and methanol has toxic effects on the human body. In contrast, butyrate exhibits a stronger specificity in its association with constipation. Consequently, we ultimately designated butyrate as the sensing biomarker for our engineered bacteria.
In the second engineering cycle, we aimed to simplify the genetic circuit to achieve better controllability and expression efficiency. However, through bacterial experiments and comparisons, we found that the existing butyrate sensing circuit in enterohemorrhagic Escherichia coli was more sensitive and efficient. Therefore, we decided to retain the existing butyrate sensing circuit of enterohemorrhagic Escherichia coli.
In the third engineering cycle, we attempted to the increasing expression of Lrp within the bacteria to enhance their sensitivity to butyrate. Through bacterial experiments, we confirmed that increasing the expression of Lrp indeed enhanced the sensitivity of our engineered bacteria to butyrate.
In the fourth engineering cycle, as our designed butyrate sensing circuit is butyrate-inducible, meaning that a certain range of high butyrate concentrations can promote the expression of this gene circuit, and since butyrate concentrations decrease during constipation, we needed to add a negative regulatory element to enable our engineered bacteria to express and secrete therapeutic substances during constipation. Through literature research, we discovered Cl/plam, where Cl is a repressor protein that can bind to the plam promoter and inhibit downstream gene expression. Subsequently, we successfully validated the effectiveness of Cl/plam negative regulation through bacterial experiments.
In the fifth engineering cycle, we attempted to use endogenous substances from the human body as the therapeutic substances expressed and secreted by our engineered bacteria. Through literature searches, we found that 5-HT can effectively promote intestinal motility and secretion. Therefore, we planned to have the engineered bacteria express the two key enzymes during the 5-HT synthesis process: TDC and TPH, utilizing tryptophan in the intestines to synthesize 5-HT. We successfully expressed these two enzymes in our engineered bacteria through subsequent bacterial experiments.
Cycle1:Research
How do our engineered bacteria perceive the state of constipation in the body?
Constipation is a relatively common phenomenon, and unhealthy lifestyles, as well as bad eating habits, can lead to constipation. According to statistics, the prevalence of chronic constipation in Chinese adults is between 4% to 6%, increasing with age and reaching as high as 22% in the population over 60 years old. Currently, there are several main therapies for constipation, including medication, dietary adjustments, biofeedback therapy and surgical treatment. However, these treatments face challenges such as short-term efficacy, significant trauma, and potential side effects. There is an urgent need to explore a new treatment approach. We aim to leverage synthetic biology principles to create an engineered bacteria capable of detecting the body's constipation state and then secreting relevant substances to alleviate constipation.
Through research and investigation, we have found that when the body experiences constipation, there are changes in the concentrations of methane gas and butyrate within the intestinal tract.
Relevant experiments indicated a significant association between methane excretion and chronic constipation. In a glucose breath test (GBT) conducted on 50 patients experiencing gastrointestinal symptoms, it was found that constipated individuals had a higher median CH4 excretion compared to those with regular daily bowel movements (p=0.0406). Constipated patients exhibited an average CH4 emission of 30.3 ppm, while the average CH4 excretion in the normally defecating population was 21.5 ppm. It can be concluded that when the body experiences constipation, there is an elevated concentration of methane gas within the intestinal tract[1].
Short-chain fatty acids (SCFAs), with butyrate making up approximately 60% of them, are primarily produced through fermentation and degradation by the gut microbiota in the human intestinal tract. It is estimated that the concentration of butyrate within the intestinal lumen of humans and animals ranges from 10 to 20 mM. When the body experiences constipation, there is a shift in the gut microbiota, resulting in reduced butyrate production. During constipation, the concentration of butyrate within the intestinal tract can drop below 10 mM. It can be concluded that the concentration of butyrate in the intestinal tract decreases when constipation occurs[2].
Cycle1:Imagine
Can engineered bacteria detect the state of constipation in the body by sensing changes in methane or butyrate concentrations?
Cycle1:Design and Build
The AOX1 promoter is a commonly used high-efficiency inducible promoter in Pichia pastoris yeast. When methanol is the sole carbon source, the AOX1 promoter can be induced by methanol to initiate the expression of alcohol oxidase enzymes, allowing for metabolic processes utilizing methanol. Particulate methane monooxygenase (pMMO) is capable of converting methane into methanol. Therefore, we utilize this enzyme to convert methane within the intestinal tract into methanol. Subsequently, by inducing the expression of relevant genes downstream of the AOX1 promoter, we achieved the engineered bacteria's ability to sense methane[3].
The PchA promoter is an inducible promoter found in enterohemorrhagic Escherichia coli (EHEC). When butyrate binds to Lrp, they form a complex that subsequently binds to the PchA promoter, initiating the expression of PchA. PchA then interacts with the Lee promoter, thereby promoting the expression of genes downstream of the Lee promoter. Therefore, our goal is to introduce the PchA promoter into the engineered bacteria, enabling them to sense butyrate[4].
Cycle1:Test
Through HP investigations, literature reviews, and group discussions, the production and emission of methane gas in the intestines are associated with various factors, including the types and quantities of intestinal bacteria, dietary habits, and intestinal motility. While methane gas is correlated with constipation in some cases, it is not always accompanied by elevated methane gas levels in all instances of constipation. An increase in methane levels may be a normal physiological phenomenon, such as with a high-fiber diet or active intestinal peristalsis. Therefore, the specificity of the association between methane and constipation is not strong. Additionally, we discovered that methanol is a colorless, volatile liquid. When ingested by the human body, it is metabolized into formic acid and carbon dioxide, both of which enter the body and can lead to poisoning. Methanol has varying degrees of toxic effects on the central nervous system, cardiovascular system, digestive system, and visual system of the human body. In contrast, butyrate demonstrates a stronger specificity in its association with constipation. Under normal circumstances, the concentration of butyrate in the intestines remains within the range of 10-20 mM, but during constipation, the concentration of butyrate drops below 10 mM.
Cycle1:Learn
Therefore, we ultimately chose to use butyrate as the sensing marker for our engineered bacteria.
Cycle2:Research
Under the professional guidance and advice of our teachers, we have made diligent efforts to simplify the genetic circuit in order to better optimize gene expression and reduce unnecessary nonspecific reactions, thereby effectively enhancing the controllability of gene expression. This process is not only aimed at achieving higher expression efficiency but also contributes to ensuring more precise control of gene expression levels under different conditions, ultimately providing more reliable results for our research or applications.
Cycle2:Imagine
Considering that the pchA promoter can sense the complex of Lrp and butyrate, initiating the expression of the pchA gene, is it possible for us to further simplify the genetic circuit? In other words, can we directly connect the protein we want to express after the pchA promoter without the need for additional regulatory elements? Such simplification could potentially enhance the efficiency and controllability of the gene expression system, providing greater convenience and feasibility for our research or applications. This concept holds promise for optimizing the gene expression system, making it more efficient and flexible, while reducing the complexity of building genetic circuits.
Cycle2:Design and Build
Designing two gene fragments: one directly attaching EGFP downstream of the pchA promoter, and the other appending EGFP after the existing gene segment in enterohemorrhagic E. coli. We compared the responsiveness and feedback levels of these two fragments to butyrate.
Cycle2:Test
Bacterial Experiment: We cloned the synthesized A segment and B segment onto the pet-28a plasmid. Subsequently, we individually transformed these plasmids into Escherichia coli BL21. After plating and overnight incubation, we extracted an equal volume of bacterial culture and inoculate it into a 96-well plate containing LB medium and sodium butyrate. Then the plate was incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.
Cycle2:Learn
E. coli strains transfected with either A segment or B segment were cultivated in the presence of 10mM and 20mM sodium butyrate. The results revealed that the sensitivity of A segment to sodium butyrate and its ability to enhance the expression of downstream genes were superior to those of B segment.
In the end, we opted for the design of the A segment.
Cycle3:Research
Leucine responsive regulatory protein (Lrp) is a crucial transcriptional regulatory factor in prokaryotes, widely expressed throughout the prokaryotic world. Lrp can function as a global or local transcriptional regulator, participating in the control of various essential physiological processes in microorganisms, including amino acid metabolism, flagellum formation, heavy metal transport, peptide transport, energy metabolism, and more. It also serves as a "feast/famine" regulatory protein, assisting microorganisms in adapting to diverse external environments[4].
As a sensor, Lrp transduces the stimulus of butyrate into the regulatory network controlling the LEE genes through the PchA-Ler system.
Cycle3:Imagine
Is it possible for us to enhance the sensitivity and responsiveness of the PchA promoter to butyrate in engineered bacteria by increasing the expression level of the Lrp protein? By elevating the expression of Lrp, we aim to bolster the engineered bacteria's ability to detect butyrate, allowing them to quickly sense the state of constipation within the body. Subsequently, this enhanced sensitivity could trigger the expression of downstream genes to address constipation, thus preventing potential harm caused by prolonged constipation to the body.
Cycle3:Design and Build
We simultaneously introduced the pBAD33 plasmid carrying Lrp and the pet-28a plasmid containing the receptor into Escherichia coli. Bacteria that received only the receptor plasmid but not the Lrp plasmid were used as a control group. We compared the sensitivity of these two groups to butyrate sensing.
Cycle3:Test
Bacterial Experiment: The experiment was divided into two groups. In one group, the pBAD33 plasmid carrying the Lrp gene and the pet-28a plasmid containing the PpchA-pchA-Plee-EGFP segment were simultaneously introduced into Escherichia coli BL21. In the other group, only the pet-28a plasmid containing the PpchA-pchA-Plee-EGFP segment was introduced into Escherichia coli BL21. After plating and overnight incubation, an equal volume of bacterial culture was extracted and inoculated into a 96-well plate containing LB medium and sodium butyrate. Each well was supplemented with arabitol to a final concentration of 3.2 mM. The plate was then incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.
Cycle3:Learn
Both groups were cultured in 0mM, 10mM, and 20mM sodium butyrate, and the results demonstrated that increased expression of Lrp does indeed enhance the sensitivity of the PchA promoter to butyrate .
Cycle4:Research
According to the literature, it is evident that the concentration of butyrate in the intestinal tract significantly decreases during constipation. Our PpchA-pchA-Plee-gene, on the other hand, is butyrate-inducible, meaning that higher concentrations of butyrate within a certain range can enhance the expression of this gene circuit. Therefore, we envision utilizing changes in butyrate concentrations to negatively regulate the products of our genetic circuit. To achieve this, our genetic circuit requires a negative regulatory element. This would enable us to promote the expression of relevant genes when butyrate concentrations decrease.
Cycle4:Imagine
Is it possible for us to consider introducing a repressor protein to achieve negative regulation of the gene circuit? By introducing this repressor protein, we aim to suppress gene expression when the body is in a normal state and, conversely, promote gene expression when the body is in a constipated state. This would enable our engineered bacteria to switch their function between normal and constipated states, allowing for a dynamic response to the body's condition.
Cycle4:Design and Build
Plam is a potent promoter found in the lambda bacteriophage, while Cl is an inhibitory protein that can bind to the Plam promoter, thereby repressing downstream gene expression. Cl/Plam is a well-known component of a genetic circuit involving a "NOT" logic gate[5].
Cycle4:Test
The synthesized PpchA-pchA-Plee-cl-plam-EGFP gene fragment was cloned into the pet-28a plasmid. Subsequently, it was co-transformed into Escherichia coli BL21 along with the pBAD33 plasmid carrying the Lrp gene. After plating and overnight incubation, an equal volume of bacterial culture was extracted and inoculated into a 96-well plate containing LB medium and sodium butyrate. Each well was supplemented with arabitol to a final concentration of 3.2 mM. The plate was then incubated at 37°C with shaking in a microplate reader, simultaneously measuring fluorescence intensity and bacterial growth curve.
Cycle4:Learn
The results indicate that at a concentration of 0mM sodium butyrate, EGFP exhibits high expression, whereas at a concentration of 20mM sodium butyrate, EGFP is hardly expressed. This suggests that the Cl/Plam element effectively serves as a negative regulatory component.
Cycle5:Research
Based on interviews and research, naturally occurring substances produced within the body possess the following potential advantages as therapeutic agents compared to chemically synthesized drugs.
Biocompatibility: Naturally occurring substances produced within the body are often more readily accepted by the human body due to their closer alignment with the body's biochemical processes. This reduces the risk of triggering severe side effects or immune reactions.
Lower Toxicity: Naturally occurring substances typically exhibit lower toxicity as they are widespread in nature and interact with biological systems. In contrast, some chemically synthesized drugs may require more chemical processing to ensure their safety.
Bioavailability: Naturally occurring substances produced within the body are often more readily absorbed and utilized because they are closely aligned with the body's metabolic pathways. This can enhance the bioavailability of drugs, reducing the required dosage and frequency of treatment.
Reduced Allergic Reactions: Naturally occurring substances are generally less likely to trigger allergic reactions, as the body has already developed a certain degree of immune tolerance towards them. In contrast, certain components in synthetic drugs may lead to allergic reactions in some patients.
Cycle5:Imagine
Is it possible to enable our engineered bacteria to produce endogenous substances when they detect constipation in the body, which can be used to treat constipation? By harnessing engineered bacteria, we can design a gene circuit with negative regulation, ensuring the release of the necessary bioactive substances only when the body is experiencing constipation. This strategy not only helps us achieve more precise treatment but also minimizes interference with and side effects on the human body to the greatest extent possible.
Cycle5:Design and Build
Serotonin (5-HT) is a vital neurotransmitter that plays a crucial role in regulating emotions, memory, appetite, gut homeostasis, and metabolism in the body. While serotonin plays a key role in the central nervous system, the majority of serotonin (up to 95%) is produced by enterochromaffin cells (EC cells) in the gut. Research suggests that the stimulation of 5-HT in the gastrointestinal tract can activate either the 5-HT2 receptors in smooth muscles or the 5-HT4 receptors in enteric neurons, leading to gastrointestinal smooth muscle contractions and promoting intestinal motility and secretion. As a result, it is widely used in the treatment of gastrointestinal disorders[6].
TDC and TPH are two crucial enzymes in the synthesis of 5-HT. They use tryptophan as a precursor, and through the action of tryptophan hydroxylase (TPH) and serotonin decarboxylase (TDC), produce serotonin (5-HT)[7].
Cycle5:Test
The synthesized fragments mentioned above were ligated into the pet-28a plasmid, which was subsequently introduced into Escherichia coli BL21. After plating and overnight incubation, bacterial cultures were collected and grown in liquid LB medium for 16 hours. The expressed proteins were then subjected to Western blot (WB) analysis.
Cycle5:Learn
Escherichia coli BL21 can effectively express both TPH and TDC enzymes.
- Furnari M, Savarino E, Bruzzone L, Moscatelli A, Gemignani L, Giannini EG, et al. Reassessment of the role of methane production between irritable bowel syndrome and functional constipation. J Gastrointestin Liver Dis. 2012;21(2):157-63. Epub 2012/06/22. PubMed PMID: 22720304.
- Koo CW, Tucci FJ, He Y, Rosenzweig AC. Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer. Science. 2022;375(6586):1287-91. Epub 2022/03/18. doi: 10.1126/science.abm3282. PubMed PMID: 35298269; PubMed Central PMCID: PMCPMC9357287.
- 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.
- Salvi PS, Cowles RA. Butyrate and the Intestinal Epithelium: Modulation of Proliferation and Inflammation in Homeostasis and Disease. Cells. 2021;10(7). Epub 2021/08/08. doi: 10.3390/cells10071775. PubMed PMID: 34359944; PubMed Central PMCID: PMCPMC8304699.
- 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.
- 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.
- 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.
Abstract
In the field of therapeutic synthetic biology, safety also involves designing measures to prevent engineered cells from harming host cells and environment. Alongside conventional methods like sterilization, the kill-switch circuit has been widely used as the first choice by previous researchers and iGEMers.
In our project, engineered ECN producing 5-HT were utilized to alleviate constipation, sensing changes in butyrate concentration in the intestine to release 5-HT, aiming to provide long-term preventive effects for individuals prone to constipation. In the four generations of our safety design, which will be mentioned later, we have considered the following concerns:
1.The uncontrolled proliferation of engineered 5-HT ECNs results in excessive production of 5-HT, which disrupts intestinal physiology, even leading to alterations in the microbial composition.
2.Engineered 5-HT ECNs have the ability to survive and replicate for a certain time outside the body after being excreted in feces, which raises concerns about potential environmental hazards, especially in aquatic environments.
3.The efficacy of engineered 5-HT ECNs is compromised as they are significantly depleted by gastric juices, preventing the desired effect from being achieved.
In our initial engineering cycle, we considered environmental protection first and foremost. So we added a temperature-controlled kill switch to ensure that the engineered bacteria will die after being expelled from the body. Then when conducting questionaries to the general public, we found that people were afraid of the bacteria and questioned the safety of the engineered bacteria in vivo. At the same time, Prof. Deng also pointed out the necessity of adding an in vivo containment system.
In our second engineering cycle, we followed Prof. Deng's suggestion, and after absorbing the experience of our previous teams, we designed a short and compact dual-drug containment system and conducted relevant proof-of-concept experiments with preliminary results. In a new round of public research, we found that yogurt, as a laxative food, was favored by the elderly, who preferred yogurt rather than capsules to be used as a carrier for the engineered bacteria.
So, in our third engineering cycle, considering the complex transportation and preservation conditions of yogurt, we specifically designed the kill switch and anti-acid encapsulation for yogurt to adapt to various scenarios from transportation to excretion. Although not ready for experimentation, the skeleton of our route has been fully characterized by the original article. At the same time, it is worth mentioning that, adhering to the principle of local people solving local problems, we do not have a one-size-fits-all approach to the positioning of our product carriers, as well as the product itself, and in the future, we will design our product in a comprehensive manner, taking into account the policy and geographic characteristics of the area.
In our fourth engineering cycle, at PI's suggestion, we made a simple design for quorum sensing. In our design, the colony is only allowed to grow rapidly when both the colony density and butyrate concentration are below the threshold, which greatly ensures in vivo safety. We will validate this part of the design in the future.
Cycle1:Research
Kill-switch for environmental protection
In our group discussion, we firstly considered the need to exterminate engineered bacteria upon their release into the environment thus preventing potential contamination in future application scenarios, namely, a kill-switch circuit for environmental protection.
Despite the powerful sterilization capacity of conventional physical and chemical methods, they are inconvenient to use and may pose risks of accidental harm to users, making the kill-switch circuit a better choice[1]. (Detailed information can be found on our Safety page).
We conducted research on previous teams' kill-switch strategies, and common circuits found include temperature control, oxygen control, light control, and pH control, all based on inherent differences between the internal and external environments.
Cycle1:Imagine
Temperature control strategy
We decided to choose the currently maturest strategy, temperature control strategy. We envisioned that at 37℃ in the human body, bacteria can grow normally. However, when they are discharged into the sewage system and the temperature drops below 30℃, the expression of toxic proteins (such as mazF, ccdB, rel, etc.) was elevated, leading to extensive bacterial death[2,3].
Cycle1:Design and Build
PCspA-mazF as our circuit
The cold-acting promoter CspA can only be efficiently translated at a low temperature[4], thereby inhibiting the expression of downstream genes, namely toxin proteins in our design. We made a comparison between the most commonly used toxic proteins, mazF, ccdB, relE, finding that both mazF and ccdB are suitable while relE have the potential to cause cell death in human cell lines according to team Fudan_2020 on part BBa_K185047[5]. We finally chose mazF as our toxic protein considering patent issue of ccdB.
Cycle1:Test
Questionnaire survey
Our HPers conducted a questionnaire survey mainly targeting the elderly to explore their acceptance of our engineered bacteria. Unfortunately, the survey results were discouraging as the elderly expressed concerns regarding the safety of the bacteria.
Cycle1:Learn
Inducible containment system
During the discussion with Professor Deng Zixin, an expert in the field of microbial metabolism, we discovered a flaw in our safety module, the lack of consideration for urgent containment within the body. Additionally, as mentioned before, our HPers conducted a questionnaire survey among the elderly, revealing that they harbor preconceived notions about bacteria and lack sufficient understanding of engineered bacteria, leading to concerns regarding the use of engineered bacteria for constipation treatment.
To overcome this obstacle, it is essential not only for the HP group to promote and educate about probiotics but also for us to incorporate an inducible containment system for probiotics. Having a self-controlled system will instill a stronger sense of security among individuals.
Cycle1:Improve
Drug control strategy
We decided to introduce a more flexible and autonomous system, drug control system, widely used in in-vivo containment system, and conducted research and exploration.
Cycle2:Research
In-vivo urgent containment
In response to the concerns regarding the in-vivo safety of bacteria, we conceived the idea of designing an in-vivo containment system by introducing drug control system. With the advice from Professor Deng Zixin, an expert in the field of microbiology, who suggested incorporating an emergency containment for our engineered bacteria, we became more determined in pursuing this idea, collecting and screening drug control strategies.
Cycle2:Imagine
Ideas of double drug control
Common drug control circuits are designed to be induced by rhamnose, arabinose, and tetracycline. While browsing through previous wikis, we came across an intriguing switch called the l-malic acid switch[6,7,8]. It offers a better taste flavor compared to rhamnose and arabinose, making it more readily accepted. We planned to utilize the l-malic acid-sensing switch to induce bacteria death, alongside the rhamnose switch for environmentally exposed death, which constitute a double drug control system.
In practical application scenarios, it would be necessary to take an appropriate amount of rhamnose to maintain a certain concentration in the intestine, ensuring normal bacterial growth. When it becomes necessary to stop the treatment, l-malic acid intake can be utilized to halt the growth of engineered bacteria within the body.
Cycle2:Design and Build
L-malic acid and rhamnose to constitute double drug control
The double drug control strategy is implemented through the induction of rhamnose and l-malic acid. In an environment lacking rhamnose, the engineered bacteria will not express the tetR, and the ptet promoter activates the expression of the toxic protein mazF, thereby triggering apoptosis, which makes rhamnose a necessary addition for their survival. The l-malic acid switch is activated by l-malic acid and initiates the expression of downstream toxic proteins, allowing patients to independently eradicate the bacteria in their bodies by taking l-malic acid.
Cycle2:Test
Bacterial experiment|Verification of the circuit pRha-tetR-ptet-mazF
Rhamnose kill-switch is for environment protection, that is to say, when bacteria is excreted outside and rhamnose is diluted, bacteria will die due to the kill-switch, which is triggered by low concentration of rhamnose.
Pre-experimentation via plate spreading showed that rhamnose addition or not had a significant effect on the survival of bacterial and subsequent OD evaluation under different concentration of rhamnose co-culture for 36 hrs showed that groups with higher concentration of rhamnose grew better, which met our expectations.
a.Pre-experimental plate spreading results, with rhamnose addition or not
b.Visualization of (a)
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of rhamnose
Bacterial experiment|Verification of the circuit pT7-mleR-p_mleS-mazF
Malic acid kill-switch is for in-vivo urgent containment, which means the patient can control the process as their wish by taking in some malic acid with good flavor.
Pre-experimentation via zone of inhibition showed that malic acid addition could inhibit the bacteria growth. Then we carried out OD evaluation, which was unsatisfactory for OD evaluation could not distinct living and dead individuals well. So we performed interval plate spreading at 2h, 4h, 6h, 8h, 10h, 12h under different concentrations of malic acid co-culture, which showed that malic acid addition could exert killing effect.
a.Anti-bacterium-circles testing as a pre-experimentation, different concentrations of malic acid treatment
b.Visualization of (a).
c.Growth curve measured by enzyme-labeled Instrument, co-cultured for 36 hrs with different concentrations of malic acid
d.Interval plate spreading counting results and visualization
Cycle2:Learn
Deficiencies of current system in future use
Our experimental results demonstrate the feasibility of the circuit. However, we still found two deficiencies if put into practice. Firstly, l-malic acid also presents in many fruits, which may accidentally trigger the kill-switch after eating abundant fruits. Further investigation is needed to compare the l-malic acid sensing threshold with the concentration of l-malic acid in the intestines after consuming abundant fruits to determine the viability of this strategy.
Secondly, in addition to the engineered bacteria, the intestines harbor a vast population of other microbial communities. Currently, it is uncertain how these communities may affect the concentration of rhamnose. Furthermore, the complex environment of the intestines could potentially lead to rapid depletion of rhamnose to a lower concentration than the threshold required to maintain the activity of the engineered bacteria. One possible solution is to incorporate a positive feedback switch, which would allow even lower concentrations of rhamnose to sustain microbial activity. Moreover, another questionnaire survey carried out by our HPers showed that yogurt as the carrier was more preferred than capsules among the elderly, considering that products like yogurt demand higher transportation and preservation conditions, we may design a yogurt specialized kill-switch circuit.
Cycle2:Improve
Yogurt specialized kill-switch circuit
We redesigned the a yogurt specialized kill-switch circuit, well adapt to transportation and preservation conditions.
Cycle3:Research
Yogurt: a preferred delivery methods among the elderly
Another questionnaire survey targeting the elderly was conducted. The survey specifically compared two delivery methods, yogurt and capsules. The results indicated that the elderly preferred the form of yogurt. We then had a discussion concerning above results with biomedical expert Liao Xiang . He held the points that both yogurt and capsules can be used as carriers for engineered bacteria, but if yogurt is used, the packages need to be designed to withstand stomach acid and transportation issues need to be considered.
Therefore, we explored new kill-switch circuit specialized for yogurt and acid resistant coats[10] for bacteria, for yogurt can not be made into an enteric form like capsules, which can been found in our cycle of coats. Due to the excessive contents in this cycle, we've listed cycle of coats in the supplementary pdf Cycle of coats.
Note: The iGEM spirit emphasizes "local people solve local problems". Considering the vast territory of our country, transportation cost is a crucial factor to consider when designing a product. Specifically, yogurt has strict requirements for transportation conditions, and the high cost of cold chain transportation poses a challenge to nationwide promotion of our product. Therefore, we have not completely abandoned the capsule carrier. Additionally, due to its lower transportation cost, it may be suitable for promotion in the central and western regions of our country.
Cycle3:Imagine
Kill-switch circuit specialized for yogurt
Yogurt products offer excellent flavor, but they require strict storage and transportation conditions, including refrigeration. Building upon the existing literature's framework, we have redesigned a set of inducible suicide environmental exposure switches to adapt to various scenarios: transportation, in-vivo environments, and excretion scenarios. These modifications aim to further enhance the safety of the engineered bacteria.
Cycle3:Design and Build
Different endings in different scenarios
We envisioned three scenarios for engineering bacteria carried by yogurt: transportation scenario, in-vivo scenario, and excretion scenario. Engineering bacteria should be kept alive in transportation scenario and in-vivo scenario and exterminated in excretion scenario.
Circuit specialized for yogurt
In the first route, when a small amount of tetracycline (tet) is introduced, it undergoes a conformational change upon binding to Tet repressor (tetR), causing the inhibition of Ptet to be lifted and resulting in LacI expression. This, in turn, suppresses the synthesis of tetR, further weakening the inhibition on Ptet. As a result, a positive feedback loop is formed, leading to the high expression of mazF. Therefore, only a minimal intake of tet is required to exert strong repression on the engineered bacteria[11,12].
In the second route, based on the study by Wang et al[13], we envisioned 3 scenarios:
Scenario One: Transportation scenario, where the temperature is below 30℃. In this scenario, Cl857 inhibits pR, resulting in no expression of mazE but expression of mazF. However, the addition of yogurt containing rhamnose allows for simultaneous expression of mazE in the engineered bacteria, preventing bacterial death.
Scenario Two: In-vivo scenario, where the temperature is 37℃, Cl857 releases the inhibition on pR, leading to the expression of phIF and suppression of downstream mazF expression. At the same time, mazE is expressed. Regardless of whether there is a sufficient concentration of rhamnose to induce mazE, the bacteria can survive.
Scenario Three: Excretion scenario, where the temperature in the sewer system is below 30℃, mazE is not expressed while mazF is expressed. Additionally, rhamnose is diluted, insufficient to induce mazE expression. As a result, the bacteria die.
Cycle3:Test
Currently, we have only conducted experimental designs for this system. Although the framework of the circuits has been well characterized in the literature and stood the test of time, we will strive to complete and refine our validation in the future. At the same time, the current design is overly complicated, so simplification is also one of the tasks we will undertake in the future.
Cycle3:Learn
Drawbacks of current system and lessons
Tetracycline, as an antibiotic, has certain side effects and may have negative impacts on other gut microbiota. Although the designed positive feedback amplification system allows for the induction of engineered bacteria death at very low concentrations of tet, exploring alternative systems is highly necessary for safety considerations. Furthermore, during our review of the previous team's wiki, we discovered that in the case of inflammatory constipation, there may be damaged microvessels in the intestine. Could the engineered bacteria potentially take advantage of this opportunity for invasion? SZU2021 has already provided a good response to this question. Meanwhile, our PI Changhai Lei raised a question that whether we can construct a Quorum Sensing Module to further ensure the safety by controlling the population density, which better reflects the preventive role of our engineered bacteria.
Cycle3:Improve
Quorum Sensing Modules
We designed a set of Quorum Sensing Modules to control the population density, making sure that only when the population and butyrate concentration are both below the threshold, can our bacteria quickly grow.
Cycle4:Research
Quorum Sensing for population control
In nature, for multicellular groups as well as single-cell populations, there is a very simple yet crucial function: cell community control. However, in unnatural environments, E. coli community populations are not so easily stabilized. And in such a special environment as the human gut, in order to ensure the homeostasis of the intestinal flora and thus the safety of our project within the dynamic range, we must add a quorum sensing module to the Gut Sweeper.
When searching for information concerning Quorum Sensing, we were impressed by the work done by professor You in 2004 published on Nature, laying the framework for our design[14].
Cycle4:Imagine
Colony density and butyrate concentration to be the indicators
We envisioned that bacterial growth capacity is strictly kept under control in two scenarios: 1) colony density exceeded threshold, 2) butyrate concentration surpassed the threshold, which means that the colony is only allowed to grow rapidly when both norms are below the threshold.
Cycle4:Design and Build
Colony density sensing module and Butyrate concentration sensing module
Our quorum sensing module is divided into two sub-modules.
1)Colony density sensing module
2)Butyrate concentration sensing module
Controlled by the two sub-modules, the colony is only allowed to grow rapidly when both norms are below the threshold
Circuit of our two sub-modules
When the population density is low, the dominant promoter expresses the downstream genes LuxI and LuxR, which express a small amount of AHL protein and LuxR protein, respectively, and due to the low concentration of AHL protein, it is not enough to bind all LuxR protein. The free LuxR protein cannot induce the luxl promoter to express tetR and bga2 proteins, and can only induce the tetR promoter to express GDH (glutamate dehydrogenase), so it does not lead to bacterial death.
When the population density is high, the dominant promoter increases the expression of the downstream gene Lux, which expresses a large amount of AHL protein. And the high concentration of AHL protein binds to LuxR and forms a dimer. This dimer has the opposite effect to the free LuxR protein, inducing the luxl promoter to express tetR and bga2 proteins, and failing to induce the tetR promoter to express GDH (Glutamate dehydrogenase), while at the same time the induced expression of the tetR protein also inhibits the expression of GDH. And GDH is a protein that must be expressed for cell growth, so inhibition of GDH expression will lead to bacterial death.
Therefore, this sub-module I can prevent the dysregulation of bacterial community homeostasis caused by excessive population density.
In normal people, the concentration of butyrate is high, which leads to the high expression of tetR downstream of PLEE. No matter how the population density, tetR’s sufficient inhibition to the promoter PtetR prevents the expression of GDH in the downstream of PtetR. Besides, the effect is more obvious when the population density is high (because tetR can also be produced in the downstream of PluxI promoter), so the bacteria can not proliferate.
In people with constipation, the concentration of butyrate is low, so the amount of tetR expression is also low. If the population density is high, the PluxI promoter will be active, so enough tetR still can be produced, then bga2 expresses but GDH is suppressed. Only when butyrate and population density are both low that makes PtetR active due to insufficient inhibition of tetR production. Then the expression of downstream GDH is promoted and the number of bacteria grows.
Cycle4:Test
Quorum sensing based on the AHL system has been widely used in synthetic biology since its proof-of-concept by You et al. in 2004. As for the butyrate sensing backbone and tetR backbone in module II, we have already covered them in our previous experiments, and both of them achieved the expected results. Complete tests will be carried out in the future.
Cycle4:Learn
Colonization ability is yet to be improved
The Quorum Sensing system keeps the population under control and allows the colony to multiply rapidly in certain conditions. An important prerequisite for this system is that the colonies are able to colonize over a period of time rather than pass through the gut. In cycle3, we have designed a mucin and tannin encapsulation with reference to the literature. According to the report, bacteria can colonize for several days after encapsulation[10]. So such an encapsulation can be used in conjunction with a Quorum Sensing system to achieve colonization and population control simultaneously over a period of time. However, how to further extend the colonization time to better demonstrate the constipation preventive role of our engineered bacteria is one of the directions of our future work.
Cycle4:Improve
Summary of our design
In the above four cycles, with the support from human practice, we envisioned and designed double drug kill-switch, yogurt specialized kill-switch, encapsulation for acid resistance and Quorum Sensing system, but most of the tests are still at the stage of literature reference, and more experiments are yet to be carried out.
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