HP
1.We went to the nursing home near the school to carry out voluntary activities to learn from the spirit of Lei Feng. During the communication with the elderly, we learned that many elderly are plagued by constipation. So we thought about whether we could use synthetic biology to solve the problem of constipation to benefit the elderly. We first needed a wider sample of data to clarify how many people in the elderly population had constipation.
2.We reviewed a large number of publicly available data on constipation in various provinces and cities in China, and designed a questionnaire to publish online.
3.Moreover, we conducted research and interviews in many provinces and cities across the country, and found that the proportion of constipation in the elderly is quite high, which brings them great pain and increases their risk of developing other diseases. We are more deeply aware that constipation is a very important problem to be solved.
Wet Lab
1.We learned some experimental protocols and basic theories of synthetic biology genetic circuits.
2.We are trained in some basic experimental operations, like plasmid construction, cell culture, SDS page, and western blotting.
3.We discussed a pivotal question concerning our project: how engineering bacteria can perceive that the body is in a state of constipation. So we conducted a review of the literature to understand the relevant aspects.
Safety
Learn from what previous teams did to ensure safety through out the project and gain experience.
Model
Learn from previous mathematical modeling work and gain experience.
HP
1.Next, we interviewed Traditional Chinese medicine teacher to learn about the concept of "treating diseases before they occur" in traditional Chinese medicine, which inspired our project: perhaps we can use synthetic biology technology to design a drug to prevent people from developing constipation, rather than waiting until the patient already has a constipation problem.
2.During the communication with teammates in the wet lab, they suggested that they might utilize E. coli as chassis to design a living bacteria drug, which can colonize in the intestine for long after being taken in. Considering that our original design was to effectively solve constipation problems among the elderly, we went to the community again to interview the elderly to obatain their opinions on our project. We found that they concerned a lot about the safety of engineered bacteria. They also conveyed that as long as safety is guaranteed (such as national recognition), they are willing to accept this kind of living bacteria drug that can prevent and solve constipation problems in the long term. In addition, regarding the pharmaceutical form, they preferred food products such as yogurt rather than conventional forms such as capsules.
3.Since the wet lab was puzzled to determine the initial sensing substances between methane and butyrate, we contacted Professor Wang Lianghua, director of the biochemistry teaching and research department of our school, to ask about the selection of constipation markers and whether the orthogonality and robustness of the current circuit design are appropriate. He proposed that the specificity of methane as a receptor may not be strong and suggested that we try to simplify the genetic circuit to make the gene expression more efficient.
Safety
1.We began to explore the design of kill switch circuits, looking for triggers and effectors via literature review.
2.The first generation of kill switch circuit was designed, consisting of a temperature control promoter and toxin protein MazF, to achieve the purpose of environmental protection.
Wet Lab
1.The promoter AOX1 that senses methane changes and the promoter pchA that senses butyrate changes were screened from the literature, and it is intended to use two promoters to preliminarily design genetic circuits.
2.Two genetic circuits were designed. The first one is to sense methane changes, which needs to use granular methane monooxygenase (pMMO) to convert methane into methanol first, and then methanol acts on pAOX1, thus initiating expression. The second one is to sense the change of butyrate, which binds to Lrp protein, acts on PpchA, and then initiates downstream gene expression. However, our engineered bacteria cannot feel these two substances at the same time, so we can only select one as a marker of our engineered bacteria feeling constipation.
3.We reviewed the literature to see which is more appropriate as a marker of constipation in our engineered bacteria.
HP
1.Through interviews with community residents, we learned that they concerned a lot about the safety of engineered bacteria. In order to obtain professional opinions on the safety of engineering bacteria, including the relevant principles and policy, we interviewed Professor Deng Zixin, an expert in the field of microbial metabolism. He suggested to us that we need to design a safety module from two aspects: on the one hand, we need to consider the environmental protection after engineered bacteria are excreted from the human body; on the other hand, considering the safety of engineering bacteria in vivo, we should add an in vivo inducible containment switch.
2.In previous surveys, we learned that older people in the community preferred to use probiotic products in the forms of foods such as yogurt, rather than the conventional form of capsules and other common medicines. In order to find out whether our project is possible in the current biomedical industry, and get a professional view, we consulted Mr. Liao Xiang, who focuses on investment in the biomedical industry. He proposed that both yogurt and capsules could be used as carriers of engineered bacteria, but if yogurt was used, it would require a special design to resist stomach acid and consider high demands on transportation. In addition, food and drugs have different regulatory requirements, so it is recommended to clarify the product positioning.
Safety
1.After receiving feedback from the HPers on Professor Deng's suggestions to improve the safety module, we began to review the strategies of the previous team, both in environmental protection and internal containment.
2.Based on the review, a highly operable dual drug control system was selected. The two drug control systems were preliminarily designed: malic acid killing system and rhamnose maintenance system. The effect concentration and induction conditions of the two inducers were then reviewed in the literature to determine the feasibility.
Wet Lab
1.Through literature investigation and expert consultation, it was found that although methane gas is related to constipation in some cases, not all constipation is accompanied by high methane gas levels, and elevated methane levels may be a normal physiological phenomenon, such as high-fiber diet, active intestinal peristalsis, etc. That is to say, the association between methane and constipation is not specific. We also found that methanol is harmful to the human body and causes poisoning after entering the body. The association between butyrate and constipation is strong, under normal circumstances, the concentration of butyrate in the intestine is maintained at 10-20mM, and when constipation occurs, the concentration of butyrate will be less than 10mM. Therefore, we ended up determining butyrate as the sensory marker for our engineered bacteria.
2.We tried to simplify the genetic circuit to pursue the efficiency and controllability of expression, and designed two genetic circuits, intending to compare the perception and feedback of butyrate between the two designs through bacterial experiments.
3.We searched for the fragment sequence we need in vector builder and NCBI, designed it on snapgene, and submitted it to Azenta Life Science for synthesis.
Model
Mendelian randomization was used to model and analyze whether E. coli was suitable as chassis.
HP
1.In order to understand whether our products belong to drugs, health products or food from a legal perspective, so that we can position Gut-sweeper's products and promote them more rigorously, we consulted Mr. Zhang Chengzhong, who engaged in food and drug administrative in the drug supervision and inspection department. He suggested that after passing the safety assessment, our final products should choose the appropriate declaration route according to the scope of application to determine whether the product ultimately belongs to medicine, health products or food.
2.In order to have a more comprehensive understanding of the clinical treatment of constipation, as well as the views and suggestions of front-line doctors on our program, we interviewed Dr. Yu from the Department of Rectal Surgery of our affiliated hospital. He proposed that our color module may affect the doctor's examination of the patient's feces, and at the same time, constipation will develop into some severe symptoms of the gastrointestinal tract in the later stage, and in more severe cases, there will be water-electrolyte imbalance and intestinal obstruction and even life-threatening. Therefore, our engineered bacteria need to feel the state of constipation in a short time and respond in time.
Wet Lab
1.After the gene fragment is in place, we introduce it into BL21 and culture it in LB culture medium and LB culture medium containing sodium butyrate, respectively, in a microplate reader with shaking at 37°C, while measuring fluorescence intensity and bacterial growth curve with a microplate reader. It was found that the original gene fragment of Enterohaemorrhagic E. coli had a better perception and feedback of butyrate than our simplified gene line, and finally we decided to follow the existing butyrate receptor network in Enterohaemorrhagic E. coli.
2.Since the leucine response regulatory protein Lrp acts as a sensor to transduce the stimulation of butyrate into the PchA-Ler regulatory network that controls the LEE gene, we hypothesized whether the PchA promoter's ability to sense and respond to butyrate could be enhanced by increasing the expression level of Lrp protein in engineered bacteria, and decided to test this hypothesis by increasing the expression of Lrp in bacteria.
3.We found the fragment sequences we need in vector builder and NCBI, designed them on snapgene, and submitted them to Azenta Life Sciences for synthesis.
Model
According to the growth rate of E. coli in the intestine and the rate of production of its products, the variables and mathematical derivation of the model were discussed.
Safety
1.A dual-drug control system plasmid was constructed and synthesized by Azenta Life Science.
2.Register and refine parts; design dual drug control system experiments.
Wet Lab
1.The sensory fragments were introduced into BL21, and the Lrp fragments and sensory fragments were simultaneously imported into BL21, and the fluorescence intensity and bacterial growth curve were measured after culture in LB culture medium and LB culture containing sodium butyrate, respectively. It was found that the increased expression of Lrp could indeed increase the sensitivity of PchA promoters to butyrate.
2.Since the concentration of intestinal butyrate is significantly reduced during constipation, the sensory fragment we designed is butyrate-inducible, that is, a certain range of high butyrate concentration can promote the expression of this gene line. Therefore, we envisaged using changes in butyrate concentration to negatively regulate the products of our gene route, for which our genetic circuit needs a negative regulatory element to promote the expression of related genes when the butyrate concentration decreases. Through the investigation of the literature, it was found that Plam is a strong promoter in lamada bacteriophages, while Cl is an inhibitory protein that binds to the Plam promoter, thereby inhibiting downstream gene expression. So we decided to use Cl/Plam for negative regulation, we need to find the fragment sequence we need in vector builder and NCBI, designed it on snapgene, and submitted it to Azenta Life Sciences for gene fragment synthesis.
3.After the fragment is in place, it is introduced into BL21, gradient LB culture containing sodium butyrate, and cultured in a microplate reader at 37°C with a microplate reader while measuring fluorescence intensity and bacterial growth curve with a microplate reader. It is found that Cl/Plam elements can play a negative regulatory role.
Model
The E. coli growth model and product production model were fitted to the experimental results of the microplate reader, and the two models were tested and improved.
HP
1.The experimental group has successfully engineered the bacteria to sensitively feel the declining butyrate concentration in the intestine and enhance the expression of the Plam promoter and its downstream genes through negative regulation, and then we need to choose a therapeutic substance to improve the condition of constipation, so we interviewed Chai Hui, an expert in the field of biomedicine. She recommends that we use substances that occur naturally in the body as therapeutic drugs, rather than synthetic drugs. In addition, she reminds us of the effects of bacterial overgrowth.
Safety
1.The plasmid is delivered half a month later for extraction, transduction, identification, and the experimental design protocol is improved.
2.Carry out the experiment of dual drug control system, basically obtain the expected results, summarize the experience and shortcomings, sort out the data and visualization; receive safety form feedback, and carry out rectification and improvement.
Wet Lab
Based on the feedback from the HP group students contacting Professor Chai Hui, we hypothesized whether our engineered bacteria could produce endogenous substances in the human body when they detected constipation, which could be used to treat constipation. Through literature research, it was found that 5-HT can promote intestinal motility and secretion, and is an ideal therapeutic substance. Tryptophan hydroxylase (TPH) and serotophanate decarboxylase (TDC) are two key enzymes in 5-HT synthesis, so we wanted our engineered bacteria to produce these two enzymes. We attached two enzymes to the Plam promoter so that the engineered bacteria can produce them when they sense a decrease in butyrate concentration. We need to find the fragment sequences we need in vector builder and NCBI, designed them on snapgene, and submitted them to Azenta Life Sciences for gene fragment synthesis.
Model
According to the metabolic rate of tetracycline in the intestine and the speed of drug module control of suicide, the variables and mathematical derivation of the model were carried out, and the model fitting was carried out based on experimental data for verification and improvement.
HP
After some improvements to our project product, we determined that the final form of the product was mainly yogurt, positioned as a drug, through which the engineered bacteria were delivered into the intestines of the target population. But at the same time, considering factors such as transportation and preservation, we also retained the design of the capsule carrier in the form of the drug. In order to gain the views and attitudes of the elders after the improvement of our project, we arranged a community return visit. After the improvement of the project, the trust of the elderly in the community in the safety of our project products has increased, and they said that if our products are approved by the state in the future, they will be willing to try.
Wet Lab
Safety
1.On the basis of the skeleton provided by the literature, the new kill switch circuit specialized for yogurt was designed, the plasmid and experimental design were constructed, and the check in form feedback was received, all of which passed the review.
2.Due to the complexity of the plasmid backbone and the high reliability of the literature data, consider postponing the experiment in the future; and review and submit the final safety form.
3.Under the PI suggestion, the crowd sensing module is designed to increase safety; Preliminary text writing.
Wet Lab
BL21 was introduced into the gene fragment and liquid-cultured in LB culture medium for 16 h, and then WB detection was performed on the expressed protein. It was found that Escherichia coli BL21 could successfully express TPH and TDC enzymes.
Model
1.Write descriptive materials related to modeling, and sort out and examine the process of mathematical modeling.
2.Cooperate with the animation team to visualize the model.
1.Each group performs text organization and wiki upload.
2.All members work together to check wiki text, images, and videos for errors, including the part interface.
3.Conduct presentation video shoots and edits; prepare for the jamboree.