Safety

Overview

BUCT has always placed significant emphasis on biosecurity concerns. The probiotics we have designed for the gastrointestinal tract employ a non-toxic and harmless chassis, E. coli Nissle 1917 (EcN), which is a harmless strain of Escherichia coli with probiotic activity. Among Gram-negative microorganisms with probiotic properties, the strain of E. coli known as Nissle 1917 (EcN) is one of the most extensively researched strains today. For nearly a century, the EcN strain has been used as an active ingredient in licensed pharmaceuticals sold in Germany and several other countries.

Obviously, considering that we have genetically engineered it, to mitigate the risk of bioleakage, we have also designed corresponding kill switches to ensure that our engineered bacteria die in non-working environments, thus ensuring biosecurity.

Kill switch

The engineered bacterial strains designed by BUCT operate within the human intestinal tract. Human gut bacteria naturally enter the urban sewage system with excreta, eventually reaching centralized wastewater treatment facilities or being exposed directly to the natural environment. To address both of these scenarios, BUCT has implemented a dual safety mechanism, which consists of two components referred to as "ammonia suicide" and "oxygen suicide." These mechanisms come into play separately, with "ammonia suicide" functioning in septic tanks before the engineered bacteria reach wastewater treatment facilities and "oxygen suicide" acting in the wild environment, ensuring the termination of the bacterial strains.

1 Ammonia Suicide

In the system where Escherichia coli is used for the synthesis and mutual conversion of glutamate and glutamine, GOGAT-GS is a crucial component. Within this system, our interest was drawn to the promoter of the glutamine synthetase-encoding gene, glnA. As part of the Escherichia coli Nitrogen Regulation (NTR) system, this promoter can be activated when there is a lack of external nitrogen sources. It does so by phosphorylation through its transcription factor, ntrB, inducing the further formation of a hexameric structure with ntrC, which acts as a sigma factor for glnA, thereby enhancing the strength of this promoter.

After gaining insights into such a component and its characteristics, BUCT conducted a review of relevant literature and found no reports on the specific binding domains for glutamine and ammonium nitrogen regarding this component. Consequently, we planned to design experiments for the characterization of the binding domains of this native component.

1.1 Characterization of detection threshold of glnAp2 original promoter

Currently, in the characterization of promoter strength, the use of fluorescent reporter proteins is a highly effective method. Therefore, BUCT constructed the pZe-glnAP2-mCherry plasmid to overexpress the promoter under different external nitrogen source concentrations, observing the expression level of the fluorescent protein to determine the responsiveness of the promoter.

In the characterization of the native promoter, we aimed to investigate whether this component could respond to different nitrogen source concentrations. In this induction phase, we used its direct precursor, glutamine, for induction.

After electroporating the overexpression plasmid and performing selection, individual colonies were picked and cultured in a 96-well plate with shaking in LB medium for 12 hours. Then, the cultures were transferred to an enzyme-linked immunosorbent assay (ELISA) plate. In nitrogen-depleted medium, different concentrations of glutamine were added for induction, and continuous induction was carried out for 24 hours.

The results are as follows:

It can be observed that when the glutamine concentration reaches 800 mg/L, there is a significant decrease in promoter strength. This phenomenon is undoubtedly encouraging, as it indicates that with the increase in glutamine concentration, once the "nitrogen deficiency" state is relieved, the promoter's strength shows a noticeable decline. This is in line with expectations and confirms that the overexpressed promoter is functioning properly, providing a premise for our modifications.

However, we still found that if the original promoter was used, its expression intensity could not be significantly changed under the actual application concentration, so it was necessary to modify it.

1.2 Random mutation of glnAp2 and characterization of its indirect responsiveness to ammonia nitrogen

For the "ammonia suicide" system, it is essential that the modified Escherichia coli die in the septic tank environment. Therefore, one of the most significant environmental differences in this setting is the concentration of ammonia nitrogen.

As a result, we contacted the Beijing Municipal Wastewater Treatment Center in China with the hope of obtaining the test data for various physicochemical indicators of sewage in residential area septic tanks. Fortunately, we received a response from the Beijing Municipal Wastewater Treatment Center, which informed us of the ammonia nitrogen levels in septic tanks in Changping District, Beijing, for the year 2022. These levels ranged from 150 mg/L to 450 mg/L (with bi-monthly testing, averaging approximately 298.7 mg/L over 24 tests in 2022). Upon obtaining this data, we immediately initiated our experiments.

Using the original glnAP2 promoter sequence as a template, we performed error-prone PCR amplification using the Beyotime QuickMutation™ Gene Random Mutagenesis Kit. The resulting mutant sequence library was then homologously recombined with the mCherry fluorescent protein and the pZe plasmid backbone, creating a fusion plasmid containing the entire mutation library, referred to as pZe-glnAP2(random)-mCherry.

This plasmid was transformed into Escherichia coli Nissle 1917, which already contained the integrated 50trc-gdhA gene (details in the results section), using electroporation. Single colonies were picked and cultured in deep-well plates with LB medium. Well A1 served as the control (original). The cultures were grown at 37°C and 1000 rpm for 12 hours to create a seed culture. Subsequently, the seed culture was transferred into a 96-deep-well plate with nitrogen-free medium containing 10 g/L glucose. (The seed deep-well plate was stored at 4°C for later use.) Ammonium chloride (NH4CL) was added at the appropriate concentrations (0, 150, 300, 400 mg/L), and the cultures were incubated at 37°C and 1000 rpm for 24 hours. Fluorescence intensity was measured (excitation at 580 nm, emission at 620 nm).

Selection criteria: Groups 0 and 150 displayed normal fluorescence, while groups 300 and 450 exhibited weak fluorescence.

For each corresponding bacterial strain, plasmid DNA was extracted, and 5 μL was reserved, while the remainder was sent to Huada Company for sequencing.

In practice, random mutagenesis lacks specificity in sequence modification, often requiring extensive time and continuous screening to obtain the desired sequences. Fortunately, after a period of screening, BUCT obtained a sample, referred to as "B6 B7 C6 D5", the 4 samples that met the requirements for practical application.

Fortunately, the promoter samples after random mutation can show a significant decrease in intensity with the increase of ammonia nitrogen concentration in our actual concentration, which provides an important basis for us to build a suicide system with different ammonia nitrogen concentrations as the core.

In fact, we have to mention that we screened about 1600 samples in total, and finally found these four samples in all mutated samples, which is a considerable workload for BUCT, which is completely composed of undergraduate students. After obtaining the correct mutated samples, we sequenced them. For their actual sequences, please pay attention to the part page.

B6:

B7:

C6:

D5:

1.3 Complete design of “ammonia suicide”system

To meet our biosafety requirements, we identified the bsrG-asrG toxin-antitoxin system, specifically the Type I toxin-antitoxin system bsrG/asrG(SR4), in Bacillus subtilis strain A-5. The antitoxin, asrG, is a cis-encoded regulatory RNA that neutralizes the action of the BsrG toxin. It prevents toxin expression by promoting the degradation of toxin mRNA and inhibiting its translation.

The transcriptional regulator TtgR from Pseudomonas putida NBRC 14164 belongs to the TetR family of transcriptional repressors. It inhibits the transcription of the TtgABC operon and its own transcription, thereby regulating the efflux of harmful chemicals from bacterial cells through efflux pumps. Due to the flexibility of TtgR's ligand-binding domain, it can bind to a wide variety of structurally diverse ligands.

Therefore, we designed a gene circuit for the suicide system (as shown in the figure below). In the human intestinal tract, which is a relatively low-nitrogen environment, the expression of glnAP2 is relatively high. At this time, PttgA is inhibited by TtgR. We also considered the possibility of leakage expression, so the antitoxin of bsrG will be co-expressed, and its expression level will be much higher than that of the toxin. In this state, the bacteria can survive normally.

In the septic tank, the bacteria will be in a high-nitrogen environment, and the expression of glnAP2 will be inhibited, which will release the inhibition on PttgA. As a result, the toxin will be expressed, and the bacteria will die.

We obtained the corresponding gene sequences from the genomes of Pseudomonas putida and Bacillus subtilis (primer sequences provided in the supporting information). Using the pZe plasmid as a vector, we successfully constructed the pathway.

To validate the effectiveness of the suicide system, we transformed this plasmid into cells and cultured them on a nitrogen-free liquid medium supplemented with gradient concentrations of ammonium chloride (0, 50, 100, 150, 200, 250, 300 ,350, 400, 450 mg/L) at 37°C, 12h.

The results are as follows:

It can be found that with the increase of the concentration of ammonium chloride, the OD600 of the culture medium reached the highest at 150mg/l, and then reached the lowest at 450mg/l, which is almost completely consistent with our expectation, which also proves that the construction of our "ammonia suicide" system is successful.

2.Oxygen Suicide

Fortunately, our laboratory has a mature oxygen suicide system that can cause bacteria to die in aerobic environments. This system was also used by the BUCT 2021 team. In this module, we will continue to use this system. To prevent interference between the toxin-antitoxin systems, we employed the mazF-mazE system from Escherichia coli. We envisioned engineering bacteria to survive in a low-oxygen, lactose-containing environment and self-destruct under aerobic conditions.

The following results are from BUCT 2021, and we also have a partnership with LZU-CHINA in this part.

We placed a low-oxygen promoter, phyb, before the antitoxin gene to ensure that under aerobic conditions, Escherichia coli cannot resist the toxin and will die. In anaerobic conditions, when bacteria encounter lactose, the metabolism product of lactose, allolactose, serves as an inducer for the lac operon. It binds to the allosteric site of the repressor protein, causing a conformational change that disrupts the repressor protein's affinity for the operator gene. This prevents it from binding to the operator gene. As a result, RNA polymerase binds to the promoter and successfully transcribes the structural genes through the operator gene, expressing MazF.

Simultaneously, in the low-oxygen conditions of the intestinal environment, the phyb promoter will activate and express the MazE gene. The toxin and antitoxin counteract each other, allowing the engineered bacteria to survive. The oxygen suicide system using Escherichia coli Nissle 1917 as the carrier has already been validated, so we did not revalidate it.

In the absence of oxygen, bacteria first encounter lactose. Lactose metabolite isolactose acts as an activator for the lac operon. It binds to a specific site on the inhibitor protein, causing a conformational change that disrupts its interaction with the operator region of the manipulative gene. As a result, the inhibitor protein can no longer bind to the manipulative gene, allowing RNA polymerase to bind to the promoter. This enables the successful transcription of the structural gene, leading to the expression of MazF toxin.

Simultaneously, in the low-oxygen environment of the intestines, the phyb promoter becomes active, leading to the expression of the MazE gene. The toxins and antitoxins produced by these genes counteract each other, allowing the engineered bacteria to survive.

In the presence of oxygen, the phyb promoter is swiftly deactivated, leading to the cessation of antitoxin gene expression. Consequently, the suicidal system exclusively produces MazF toxin. When the concentration of MazF toxin surpasses a critical threshold, it triggers apoptosis, ultimately resulting in the demise of the bacteria.

Using the Gibson assembly method to assemble 5 genes, and successfully obtained the built plasmid, run glue verification as shown in the following figure, the length of about 4.7kb, in line with the plasmid length.

In the case of Nissle 1917 with the successfully introduced plasmid, cultivation took place inside a glove box. After extensive experimentation to optimize reaction conditions, it was observed that Nissle 1917 exhibited significantly slow growth in an oxygen-depleted environment, necessitating a growth period of 48-72 hours to discern distinct colonies. However, when cultivated in aerobic conditions, there were no discernible signs of engineered bacterial growth, making it challenging to confirm the functionality of the suicide system.

This suicide system ensures that when the bacteria are released into the natural environment and exposed to air, they will die.

overview

Ensuring the safety of experimental items and operators is the premise of conducting any experiment. During the whole experimental process, BUCT team members always put safety first. In the whole process of our experiment, we strictly abide by the relevant provisions of microbial experiments in the General Guidelines for Laboratory Biosafety of China, and experts supervise the whole process and help us evaluate the possible risks. Our experimental plans and contents were completed under the guidance of PI, who confirmed that there were no biosafety problems in our laboratory equipment and experimental procedures.

In addition, the materials used in our experiments are not harmful to humans, such as yeast powder, AGAR, sodium chloride, glucose and other traditional media components, which are not in the danger list. Nucleic acid dyes used in agarose gel electrophoresis are considered toxic, but our laboratory demarcates a special operating area for the use of this toxic substance, and all relevant experimental equipment is not moved from this area to ensure the safety of the experimenters. Our genetically engineered E. coli Nissle 1917 is a known E. coli probiotic that is completely harmless to humans. On top of this, we also set up sophisticated growth-limiting systems in engineered bacteria to ensure that engineered bacteria commit suicide when the beneficial cell density is exceeded.

Our laboratory is a level 1 laboratory, which is a complete standard microbiology laboratory. All the personnel who enter the laboratory to participate in the experiment must learn the laboratory safety rules uniformly taught by the school, and receive basic experimental training every semester or regularly, and then attend and pass the training examination. When we conduct experiments for the project, we will be accompanied by professional laboratory operators, and the experiments will be carried out in the national key laboratory provided by PI. We have conventional sterile operating table, bacterial incubator and shaking table, and complete molecular biology experimental equipment. The materials purchased by the laboratory must be approved by the PI before they can be purchased. Laboratory waste under high pressure steam by a unified collection and treatment.

Training

Our training content is formulated in accordance with the Law of the People's Republic of China on the Prevention and Treatment of Infectious Diseases and the Regulations on the Biosafety Management of Pathogenic Microbiology Laboratories, including but not limited to:

1. Laws and regulations related to laboratory biosafety;
2. Risk assessment of pathogenic microorganism experimental activities;
3. Laboratory facilities and equipment operation requirements;
4. Guidelines for good work practices in biosafety laboratories;
5. Guidelines for handling laboratory biohazardous substances