Team:SZU-China/Engineering

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Engineering

Overview

Iterative design is a design method based on the cyclical process of prototyping, testing, analyzing and improving products or procedures. Make changes and improvements based on the test results of the latest design iteration. This process aims to ultimately improve the quality and functionality of the design [1]. In the iGEM competition, we were very concerned about engineering thinking. In the actual product realization process, we strive to get close to the most authentic user situation to obtain relevant and accurate feedback and improve our products from different angles and degrees. Therefore, we believe that engineering success should exist in project design and experimentation and all parts of the hardware or complete platform construction. This year we mainly focused on the optimization of the expression system and hardware.

Engineering Optimization of

Expression System

At the beginning of the project, we knew that we could not use the commonly used T7 promoter. Its expression activity is very high, and almost half of the metabolic flux of the whole cell can be used to express heterologous genes. This high efficiency is not conducive to the reproduction and survival of the cell itself, contrary to our hope of colonization, and cannot be used directly by us. Commonly used commercial vectors use artificial lactose operators to control the work of this promoter. In addition, we cannot use the lactose operon in the human intestine. The main reason is that isopropyl-beta-D-thiogalactopyranoside(IPTG) has specific cytotoxicity. Secondly, food contains a lot of glucose and lactose. The former has a high instantaneous content in the stomach and intestines, which potentially affects this expression system. The latter will be digested by the human body into galactose and glucose, making the IPTG induction system challenging to be regulated by humans.

Therefore, we first chose a constitutive expression system, using the most common strong constitutive promoter J23100. Although the efficiency of this promoter is much lower than that of the T7 induced expression system, as shown in figure.1, using it to control our therapeutic elements may be able to meet long-term and reasonable drug delivery requirements.

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Fig.1 Comparison between p J23100 promoter and T7 promoter.

We used this promoter to express the four genetic elements of our concern, and the specific experimental results can be seen in RESULTS. We take the use of J23100 to drive the heterologous expression of superoxide dismutase as an example. We transferred this part into DH5a, BL21(DE3), Nissle 1917, respectively. As shown in figure.2, it can be seen from the results of enzyme activity determination that this promoter can meet our assumptions above. Especially on BL21(DE3), it exhibits solid transcriptional activity and can even meet the requirements of high-efficiency exocrine expression.

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Fig.2 A Intracellular SOD activity of Nissle 1917 after transformation. B Extracellular SOD activity of Nissle 1917 after transformation.
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Fig.2 C Intracellular SOD activity of DH5α after transformation.
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Fig,2 D Intracellular SOD activity of BL21(DE3) after transformation&nbsp.
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Fig.2 E Extracellular SOD activity of BL21(DE3) after transformation.

However, we are not satisfied with the expression results of constitutive promoters. As mentioned in DESIGN, considering that the pH of the colon of IBD patients is lower than that of ordinary people, we use the PrcfB promoter to achieve an acid response. This promoter has high transcriptional activity when ph=5, but it is almost inactivated when ph=7. Nevertheless, its response to environmental acidity involves an endogenous response system of lactic acid bacteria, which involves a critical trans-acting factor, RcfB. It needs to be combined with generalized acid to make the promoter have transcriptional activity. RcfB is an endogenous protein in the lactic acid bacteria itself, but for E. coli, We additionally transformed the pet-RR plasmid vector which expresses RcfB protein into the DH5α strain that already contains the pYHSG plasmid. We chose Escherichia coli DH5α to carry this system and use a constitutive promoter to control the expression of the RcfB gene. As shown in figure.3, we finally constructed a dual-plasmid expression system, hoping to be applied in E. coli.

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Fig.3 Acid-inducible expression system mechanism.

We continue to select the superoxide dismutase gene to verify whether the promoter can meet our assumptions. We cultured the transformed engineered bacteria in media with different pH levels to the same concentration and then tested their intracellular superoxide dismutase activity. Based on this, we can verify the difference in SOD enzyme activity expressed between constitutive and acid-inducible expression systems. It can also be verified that the dual-plasmid acid-inducible expression system itself differs in expression efficiency under different pH environments. As shown in figure.4, the results show that the expression efficiency of the double-plasmid acid-inducible promoter under each pH gradient is higher than that of the constitutive promoter. Moreover, the double-plasmid acid-inducible promoter has a higher expression efficiency under low pH environmental conditions and gradually decreases with the increase of pH. This fully demonstrates that the engineered bacteria can successfully perform their role under the acidic intestinal conditions of patients with IBD but do not initiate expression in ordinary people's neutral intestinal environment, which meets our project's expectations.

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Fig.4 SOD activity of constitutive promoter and acid-induced promoter expression system under different PH conditions.

The initial cocktail therapy refers to the combined use of three or more antiviral drugs to treat AIDS, figure.5 . It highlights the combined use of multiple factors, multiple angles and multiple drugs. Thus, we think of applying this method to the treatment of IBD. Only considering the two promoter schemes and four therapeutic modules we have proposed, we can already provide eight combinations of individual therapeutic elements, needless to say the potential therapeutic targets we have found which are recognized by the academic community.

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Fig.5 Personalized cocktail therapy
Engineering Optimization of

Excrement Collection Device

As an essential part of personalized medicine, we need to perform regular and continuous faecal whole-genome sequencing for users. Therefore, conveniently, efficiently and humanely help users obtain their faecal samples and send them to third-party sequencing agencies has become an indispensable part. We conducted a series of market research and collected massive amounts of information. On this basis, we designed a variety of schemes and carried out theoretical verification and implementation one by one.

For the faecal sampling spoon, our first-generation product hopes to design a spoon-shaped pipette tip to solve the sampling of formed/non-shaped stool simultaneously, as shown in figure.6A. However, such a design will bring a high manufacturing cost, and it is essential to ensure the cleanliness of the stool sampling device. It cannot guarantee strict sterility for the user's use environment. We need a disposable device.

Therefore, in the second-generation product, we decided to design a breakable structure for our spoon head, as shown in figure.6B, to meet the high-viscosity stool sampling situation. At the same time, the top of the device is based on the design of a disposable dropper, which can collect unformed faeces/loose through the rubber tip while collecting shaped faeces. The product adopts a one-piece moulding process, and the entire device is composed of only one component, so it has a lower cost and a higher economic benefit. The integrated moulding process also facilitates ethylene oxide sterilization and packaging operations. It passed the suction test. However, due to the design structure of the broken part of the spoon handle and the accuracy of the 3D printing, there will be situations such as failure to break and adhesion.

After talking and discussing with registered structural engineers, we decided to redesign the breaking mechanism and design the third generation of products. We increase the thickness of the upper edge of the breaking mechanism (assuming that the spoon mouth is facing upward) and reduce the thickness of the lower edge of the breaking mechanism. Therefore, when a force is applied, the pulling force is convenient to break the spoon head, and the pressure will not affect the breaking of the spoon head. When we take stool samples with higher hardness and viscosity, the stress and pressure will be concentrated on the lower edge of the breaking mechanism without being broken, as shown in figure.6C . Moreover, when we break the spoon head after the faeces sampling is completed, the stress and pulling force will also be concentrated on the lower edge of the breaking mechanism, causing it to be broken. At the same time, we use SOLIDWORKS for finite element analysis to simulate the breaking effect of the breaking mechanism within a predictable pressure range.

The completion of the third-generation product has been quite good. But at the gene-sequencing company and the Gastroenterology Department of Southern University of Science and Technology Hospital, we collected opinions from practitioners, doctors, and patients to produce the fourth generation of products. According to their suggestions, a series of constant volume experiments were carried out, the minimum amount of marking was determined, and the hot stamping process was selected instead of the embossing process to reduce the manufacturing cost, as shown in figure.6D、E.

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Fig.6 A preliminary sketch of the first generation fecal sampler&nbsp.
B Computer modeling rendergraph of the second generation fecal sampler.
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Fig.6 C Sampling and breaking off freehand sketching&nbsp. D,E thermoprint freehand sketching.

We also considered a faecal collector in addition. Our first-generation collector refers to the accordion-like design, as shown in figure.7A, to reduce the folded space of the entire device. After the actual production, we found that the product was difficult to install, and it was very inconvenient to remove and discard the product after use. Finally, we decided to use the integrated stitching of two water-soluble non-woven fabrics to make it, as shown in figure.7B. When using the device, the user only needs to open the device, tear off the two-wing cloth-patterned adhesive, stick it on both sides of the toilet, and the sampling site will automatically unfold under the action of the pulling force. After sampling, the user only needs to tear off the cloth pattern glue on both sides, put the device into the toilet and flush. The cost of the device is lower, the user is convenient to use, and the potential pollution risk is avoided. This solution can also be applied to most toilet environments, with strong concealment and strong bearing capacity.

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Fig.7 A Blueprint of the first generation sampler&nbsp. B Material object of our own fecal collector.

Finally, after the above updates, we designed the final product. To enhance the applicability and humanization of the product, the whole set of products includes a series of devices such as faecal collector, alcohol cotton sheet, activated carbon mask, disposable glove, sample storage, etc. In order to facilitate users to understand the use process of the kit, considering that our stool collection kit may be applied to people of different ages and educational backgrounds, we also shot a sampling guide video to facilitate users' independent operation.

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Fig.8 Faecal collection kit box.
Engineering Optimization of

Freeze-drying Technology

We hope our live bacteria drugs to be loaded in the form of enteric-coated capsules. We need to make live bacteria into embedded bacterial powder to ensure the resuscitation activity of the flora after entering the human body and ensure the therapeutic effect of the drug. After visiting microbial pharmaceutical companies and discussing with experts, we finally chose vacuum freeze-dried bacterial powder as our live bacterial preparation form. The bacterial powder which would be small in size after vacuum freeze-drying has excellent stability and resuscitation activity, and the preparation process of freeze-drying technology is relatively stable and mature. The freeze-drying technology can ensure that the plasmid will not lost and can be expressed after resuscitation.

However, vacuum freeze-drying requires the selection of the most suitable protective agent formula for the bacterial strain chassis so that the bacterial powder is not interfered by gastric acid, has enteric properties, and ensures the bacterial activity for administration. We are very concerned about this. In combination with our chassis strains, we use Nissle 1917 for optimization. We will prepare freeze-dried engineering bacterial powder according to the formula, store the prepared bacterial powder in a refrigerator at 4 °C, and then take it out and resuscitate at the specified time point to determine and record the survival rate of the strain. At the same time, we carried out enzyme activity determination for the resuscitated engineered bacterial powder to determine the engineered bacteria's activity after freeze-drying. Due to the particularity of the expressed substance, we only choose SOD as the target of enzyme activity determination.

Based on the opinions of the literature, we determined the formula of the initial freeze-dried protective agent: 3% sucrose, 14.25% skim milk, 3% L-sodium ascorbate. We first checked the lyophilized powder for reconstitution, and the results are shown in figure.9. It can be seen that the recovery rate can be maintained at 30%-50% within one month. Concerning the existing live bacteria preparations (10^6 CFU/g) on the market, our freeze-dried powder has three orders of magnitude higher than it. Therefore, the formula has a better protective effect.

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Fig.9 Bacterial recovery rate.

However, we are still not satisfied with the SOD enzyme activity after the lyophilized powder is reconstituted. Furthermore, we noticed that the group whose resuscitation rate was measured immediately after the completion of lyophilization would have a lower resuscitation rate. We understand that when the recovery rate of engineered bacteria is measured immediately after the freeze-drying process is completed, the engineered bacteria may be Viable but Non-culturable (VBNC). This situation occurs because, under extreme environmental pressure, bacteria have entered a state of deficient metabolic activity and do not divide but are alive and can become culturable once resuscitated. Nevertheless, from the results of our recovery, the bacteria quickly got out of this state after a week of freeze-drying, so the effect on our freeze-drying effect is not significant.

Under the guidance of Dr Song Huang, we added some prebiotics to the protective agent to enhance the activity of the strain. This resulted in our second and third-generation products. The second-generation product is to replace the 3% sucrose in the unoptimized formula with 3% oligofructose. The third-generation product is based on the unoptimized protective agent formula of freeze-dried bacterial powder, directly adding fructooligosaccharides equal to sucrose in the bacterial powder and mixing it evenly.

We carried out a gradient-concentration coating plate count. The results of the second generation are shown in figure.10A. It can be found that the recovery rate of each group has decreased to varying degrees after adding fructooligosaccharides. However, the overall recovery rate is about an order of magnitude (unit CFU/g), which has little effect on the actual administration situation. The results of the gradient-concentration-coated plate counts presented by the third-generation products are shown in figure.10B. It can be found that after the addition of fructooligosaccharides, the recovery rate of the LL-37 group decreased slightly, and the recovery rate of the other groups increased to varying degrees, and the increase rate of the BSH group was even doubled.

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Fig.10 Comparison of recovery rates among different generations of products

No matter which method is used, after adding fructooligosaccharides, the enzyme activity per unit resuscitation has been increased by more than two times, as shown in figure.11. Judging from the total enzyme activity of the bacteria, the performance of the third generation and the second generation are comparable. However, since the resuscitation of the second generation of bacteria is nearly ten times of the third generation, the performance of the enzyme activity per unit of resuscitation is not very good. In comparison, the maximum enzyme activity per unit recovery amount can reach 5.2686x10^8 CFU/mL when the third generation was directly added fructooligosaccharides with the same amount of sucrose to the bacterial powder and mixed well.

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Fig.11 Comparison of enzyme activity before and after oligosaccharides.

Therefore, we decided to adopt the third-generation formula. According to the unoptimized protective agent formula of the freeze-dried bacterial powder, we directly add fructooligosaccharides equal to sucrose in the bacterial powder mix them evenly. So far, we have completed the entire process of optimizing the formulation of the freeze-dried bacterial powder protective agent.

Reference

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