- Human Pratices
When considering gene expression, we designed four sets of schemes for verification:
1. Constitutive promoter pJ23100:
It can be used to meet long-term and suitable drug administration needs, and stable and reliable protein expression is also found in experiments.
2. Promoter PrcfB(Lactobacillus):
Acid promoter P170 was highly expressed at pH =5.5 and low or no expression at pH =7.0, which was used in IBD patients' relatively acidic colon environment. PrcfB promoter is an improved form of P170 with higher expression efficiency.
3. Promoter PrcfB+RcfB protein (E. coli):
The response of PrcfB involves an endogenous response system of lactic acid bacteria, which involves a critical trans-acting factor, RcfB, which can initiate P170 activity. Since this protein is endogenous to lactic acid bacteria, we inserted genes expressing this protein into the plasmid construction to explore whether this acid response mechanism can be effective in E. coli.
4. Construction of double plasmid system:
For E.coli, we designed a two-plasmid system, in which one plasmid is specifically responsible for the expression of the response protein RcfB, and the other plasmid with PrcfB promoter is responsible for the expression of therapeutic elements in response to acid. The efficiency of the two-plasmid system is expected to be different from that of the third protocol.
We anticipate using the superoxide dismutase gene (SOD) to verify the acid response mechanism. We will test the activity of superoxide dismutase in the cells of engineered bacteria by placing them in media with different pH and the same concentration.
It is not only difficult to construct and transform plasmids in lactic acid bacteria but also there are many factors influencing the two plasmid vectors. Therefore, the completion of these four programmes is a relatively difficult task. Unfortunately, due to limited time this year, we did not complete the verification of the four schemes. We hope to further improve the experimental design in the future and establish the optimal acid response mechanism for engineered bacteria to improve the intestinal environment of IBD patients.
In our project, engineering bacteria were constructed for heterotrophic expression of SOD. Since pH was controlled in the application liquid of enzyme-substrate in the kit for measuring enzyme activity, we did not conduct a pH-enzyme activity test. We hope to explore the influence of pH on SOD enzyme activity by improving different methods of enzyme activity measurement and adjusting different environmental pH in the future, so that we can provide the best SOD supplement plan according to the intestinal pH of IBD patients.
This year, we improved the peroxidation stress state of the intestinal tract by heterologous expression of Mn-SOD. We hope to measure the concentration of Mn metal ions in the intestinal tract of IBD patients and conduct experiments to explore the impact of Mn2+ ions on SOD enzyme activity so as to supplement Mn-SOD to achieve the expected therapeutic effect. Unfortunately, due to limited time this year, we did not complete the experience. We hope to follow up and improve our experiment.
According to the literature survey, the expression of Cu/Zn-SOD decreased in IBD patients, so we believed that the SOD protein activity might be related to Cu2+ and Zn2+ ions. We hoped to measure the changes of SOD activity expressed by engineering bacteria after adding different concentrations of metal ions Cu2+ and Zn2+ to test our idea further.
This year, we hope to express the antimicrobial peptide LL37 heterogeneously to reduce the attack of endotoxin on the human intestine. When constructing the plasmid, we added a signal peptide after LL37, hoping that the expression of LL37 could be secreted for extracellular action. Unfortunately, due to various reasons, our signal peptide validation failed to obtain effective experimental results. We hope to follow up and improve our experiment to make the effect of LL37 more significant.
In this year's experiment, the protein expressed by LL37 was too small, and due to the lack of equipment and technology, we could not perform SDS-PAGE and other detection of the expressed protein. We hope that the fusion expression of LL37 with other proteins can improve the protein size, facilitate detection and promote the secretion of LL37 to improve its effectiveness.
Due to the degradation of engineering bacteria and difficulties in protein extraction, including time constraints, we were unable to conduct further studies on the heterologous expression of biliary saline hydrolyase. In the future, we hope to further explore its enzymological properties and its relationship with the intestinal environment of IBD patients, so as to provide better theoretical support for subsequent applications.
We want to investigate the enzyme activity of BSH under different reaction time to complete the basic enzymatic characteristics verification.
We hope to explore the enzyme activity of BSH under different temperature gradients to provide a better theoretical basis by comparing with the intestinal environment in human body.
We want to explore whether the enzyme activity of BSH under different pH gradients is consistent with the intestinal environment of IBD patients and has strong adaptability to low pH.
In the experimental process, we found that there was no enzyme activity of engineering bacteria after overnight culture at 37℃, while enzyme activity could be measured under cooling culture conditions. Therefore, we speculated that inclusion bodies could be formed after the expression of BSH in EScherichia coli. Therefore, we hope to add fusion protein for expression in the future so as to increase its solubility and play a better role in the intestinal environment.
BSH hydrolyzes the conjugated bile salts (glycocholate/taurocholate) in the gastrointestinal tract, converting them into amino acids and free cholic acids, which can precipitate with cholesterol and be excreted in the stool, thereby reducing serum cholesterol levels. Further validation of BSH enzyme activities is considered in our projects to provide a variety of ideas for subsequent applications.
In the process of butyric acid detection, we continue to explore the appropriate conditions, but due to the relationship of time, we failed to carry out a complete analysis of it. In the future, we hope to further improve the experimental method of derivation to achieve a more accurate detection effect and make a linear regression equation of butyric acid, carry out precision experiments, etc., to carry out qualitative and quantitative detection of butyric acid produced by us.
Colonization time and efficiency of bacteria
The colonization of bacteria in the gut is limited and influenced by many factors. In order to achieve the ideal goal of long-term and sustained drug administration, we hope to simulate the human intestinal environment and measure the colonization time of different project engineering bacteria in different environments. On this basis, the ability of engineering bacteria colonization in experimental animals was further tested to determine the frequency of drug administration of engineering bacteria and achieve the best therapeutic effect. However, due to limited time, we did not complete this part of the experiment this year. We will follow up to further improve the feasibility and pertinence of the project.
Lactic acid bacteria
In combination with our project, we hope to colonize genetically engineered bacteria in the intestines to continue to express the therapeutic products endogenously and act on the affected area to achieve therapeutic effects. Lactic acid bacteria fit our needs well in all aspects and are ideal chassis organisms.
The project pre-selected Lactococcus Lactis MG1363 as the target gene carrier. (1) Lactococcus Lactis grows fast, has a simple metabolism, and its genome contains sufficient biological information. It is a safe-grade microorganism. (2) It does not secrete protease by itself, reducing the risk of degraded foreign engineering protein. (3) The genome is small, the protein secreted by itself is few, and the inclusion body structure like Escherichia coli will not be produced. It is also conducive to the separation and purification of foreign proteins.
However, Lactococcus Lactis is a gram-positive bacteria. It has a thick and dense, rigid cell wall, so the conventional chemical methods cannot allow foreign DNA to enter the recipient cells. For this reason, we spent nearly three months exploring the electron transformation method to help us overcome the difficulty involving the pretreatment of lactic acid bacteria (lysozyme, glycine, DTT), the growth cycle of the lactic acid bacteria and the electrotransformation parameters. After repeated experiments and verification, we successfully explored the optimal scheme for the electro conversion of lactic acid bacteria.
Unfortunately, our target gene fragments were not properly cloned in high-copy or low-copy vectors, and there were gene mutations and deletions. The situation has been going on for nearly four months, and no correct fragment clones have been obtained. Many times after optimizing the experimental plan and redoing it still failed. Thus, we have to make concessions on the choice of lactic acid bacteria chassis and switch to using E. coli Nissle 1917 (EcN1917) probiotics as an alternative. EcN1917 has the function of promoting anti-inflammatory response and improving the intestinal mucosal barrier. It is mainly used clinically to treat gastrointestinal dysfunction such as Crohn's disease and ulcerative colitis.
Of course, on the one hand, our goal for this year is far more than just a chassis of Lactococcus Lactis. Lactobacillus casei, Lactobacillus plantarum and Lactobacillus acidophilus are all in our selection. We hope to compare their colonization ability and the expression quantity of foreign genes in the intestines concurrently to find the best answer for us.
On the other hand, the acid response mechanism selected by our project refers to a set of inherent expression mechanisms of lactic acid bacteria, which we designed into the E. coli expression system. (We have verified this set of mechanisms on EcN1917 strains and proved that it is effective and feasible.) After verifying that this set of mechanisms can be expressed correctly in E. coli, we will compare lactic acid bacteria and E. coli in parallel. The sensitivity of the system to pH is different.
We hope that in the future, we will overcome the difficulties of structuring lactic acid bacteria plasmids, complete the iteration of chassis strains, and screen out strains that can efficiently express engineered drugs to achieve better therapeutic effects.
Improvement of ELISA test
This year about the ELISA test to a large extent by the restriction of time and money, at the same time, we experiment using protein because the label and purification technology in the process of problems, such as some failed to the purpose of high concentration of purified protein, this also can lead to experiment is one of the reasons for not obtain satisfactory results, and in the subsequent experiments, We hope to better improve the purification technology of the target protein, so as to eliminate more unknown factors in the interaction between protein and cells and obtain better experimental results, so as to further verify the correctness of our concept.
Flow cytometry is a multi-parameter, rapid quantitative analysis of a single cell or other biological particles by monoclonal antibody at the cellular and molecular level. It is one of the most advanced cell quantitative analysis technology. It can analyze tens of thousands of cells at high speed and can measure multiple parameters from a cell at the same time with the advantages of high speed, high precision, and good accuracy.
We hope that in the cells of the subsequent experiments, the impact of cell interactions can be deeper exploration, such as damage to cells, cell secretion of TNF alpha, IL - 6, TGF - beta, IL - 10, and other factors were analyzed, and a better interpretation of engineering bacteria, and the purpose of our expressed proteins on cells anti-inflammation of the role.
This year, due to the impact of the COVID-19 epidemic and the time limit of the project, our experiment could not be successfully promoted to mice experience level. We designed a set of preliminary mouse experiment plans, hoping to supplement the mouse experiment to improve our project and improve its feasibility and scientific nature after the experiment is further characterized and successful.
Validation of therapeutic effects of
engineered bacteria on DSS - induced IBD
The experimental scheme flow is shown in the following:
Fig.1 The procedures of mice experiment.
Experimental animals: C57BL/6J mice, 7-8 weeks, 20±1g.
Model modelling and treatment: Experimental animals were divided into groups, numbered as group A to group H, with ten animals in each cage, and fed adaptively for 7-10 days. After the adaptive feeding, 200 μ L of bacterial solution containing 2×109 cells was administered to the stomach every day. Mice were fed with 5% DSS solution instead of drinking water for seven days. After that, the drinking water was replaced with sterile water, and the feeding was continued for three days. The samples were selected for the next experiment.
The mice were randomly divided into eight groups, with ten mice in each group. Except for groups G and H, the other groups were managed according to the above modelling method.
Group G was intragastric with PBS during intragastric administration, but DSS solution was not induced, and sterile water was used as drinking water throughout the whole process. The objective of this study was to evaluate the effect of PBS gavage on the intestinal microflora of mice.
Group H was fed normally without any treatment. And group H did not need to take samples and other experiments, only with other groups to take faeces. The objective of this study was to evaluate the effect of gavage on the microflora of mice.
|Group||Intragastric administration||DSS induced|
|Group B||SOD Engineering bacteria liquid||Yes|
|Group C||BSH Engineering bacteria liquid||Yes|
|Group D||Tes4 Engineering bacteria liquid||Yes|
|Group E||LL37 Engineering bacteria liquid||Yes|
|Group F||Mixture of four kinds of engineering bacteria||Yes|
|Group H||Sterile water||No|
From day0 to day17, the weight of experimental animals was recorded daily, including the sampling day, to analyze the weight change.
(The mice need to be euthanized when they lose more than 30 per cent of their weight.)
Faeces were collected from mice, and occult blood was detected. The specific steps of the faecal blood test should be completed according to the kit instructions. The body weight loss score and disease activity index (DAI) score were calculated by using the following formulas:
NTH object mass decline fraction = (NTH object mass - NTH object mass)/NTH object mass,
DAI = (decreased body mass score + stool trait score + stool blood score) /3. The scoring rules are shown in the table below.
On day0, day1, day4, day8, day11, day14-day17, the rats in each group were scored on the disease activity index.
Faeces were collected and used for 16S sequencing to detect the changes of intestinal flora in mice.
1. The consistency analysis of the initial state of mice in the same group and the bacterial community structure during the experiment;
2. Analysis of bacterial community structure consistency among each group at the experiment starting point (DAY0);
3. Changes of bacterial community at different time points in the experiment of mice in the same group, including changes before and after intragastric administration and modelling;
4. The difference of bacterial community structure in different groups at the same time point;
5. Group A and Group H were compared to analyze the influence of gavage behaviour on bacterial community structure.
The blood of experimental animals was collected through eyeball blood collection, and serum was separated to do multi-cytokine detection.
metabolome analysis to study the effect of bacterial strains on host tissues.
HE staining were performed for cytokine analysis.
After colon measurement, half (5 mice) of the colon were taken, the faeces were cleaned and rolled up in a Swiss roll manner. The colon was fixed in 4% paraformaldehyde. Paraffin section and HE staining were performed.
Peroxidase (MPO) is an indicator of neutrophil infiltration.
The rest of the colon was placed in RNA Later solution, which could be stored at 4℃ for one month or stored at -80℃
Keep for a long time. Send to analysis company for RNA extraction and transcriptome analysis.
spleen immune cells to understand the differentiation of immune cells conditions.
In the modelling part, in order to complement the idea of the project, we used test data based on experimental data in literature and papers in our Relative Abundance Analysis Model due to the limited project time and the failure to obtain real data of the human body, Etc. In the foreseeable future, we hope to implement our project and develop a mini-program specifically to serve IBD patients. Patients can see the changes in their intestinal flora corresponding to each stool test on the mini-program, and we will also provide them with personalized adjuvants.
More, we hope to provide more information in the modelling section to perfect the experiment. Still, due to the scarcity of time and animal experiments, we do not have enough data for in vivo predictions. Through a large amount of data and experimental parameters, the modelling team will establish a Neural Network Model to predict the expression level of the engineered bacteria with transferred designed plasmid in the human body.
Furthermore, the work of the modelling team will also be combined with business analysis. Use the graph theory model to mark all stool detection institutions on the world map and show the most economical and convenient addresses of stool detection institutions for patients in the mini-program.