Project safety
Overview
Our E. coli Nissle 1917 is completely exposed to the environment in practical application. Considering that the spread of the gene E. coli Nissle 1917 may pose a threat to the natural environment, BUCT tried to build an appropriate system to reduce this risk. We have considered the method of making the strain iron uptake defective. But after communicating with others, we realized that it was difficult to create a safe iron ion environment, so we gave up this design. As a team closely communicating with us, JLU-China has given us some inspiration.
After some communication, we decided to set our E. coli Nissle 1917 not only to inhibit the growth of Malassezia by consuming fatty acids, but also to synthesize an appropriate amount of 2-phenylethyl alcohol. We decided to study the intraspecific inhibition method based on the competition dynamics model of two limited resources ratio of microorganisms through modeling, and screen the promoter PQS suitable for E. coli Nissle 1917.
The old Suicide System
We want to knock out the fes gene in our E. coli Nissle 1917 so that it could not grow in an iron-restricted environment. Specifically, when the bacteria escape to an iron-deficient environment, it would die. When using our E. coli Nissle 1917, adding some iron ions can ensure that the engineered bacteria survive only in the site of application with a high probability.
Fig.1The mechanism diagram of Iron limit switch
However, it was during an exchange with other teams that we realized that it was difficult to find the relatively mild iron chelates needed in this method, because we could not guarantee that the chelates were completely harmless to the human skin. In addition, we also consulted dermatologists about whether iron ions have a negative impact on the skin, but doctors believe that iron ions are easy to cause scalp inflammation, which may further deteriorate the scalp environment. So we decided not to take iron restrictions.
The new suicide system
When we hit a low point in designing the E. coli Nissle 1917 suicide system, JLU-China and our team analysed the respective application scenarios and engineering designs, telling each other about the current problems. JLU-China has been screening various inducible promoters and found some quorum sensing promoters (PQS) that could accurately identify the density of bacteria. More precisely, when bacteria reaches a certain precise density, the expression of toxin proteins can be initiated by this promoter to direct apoptosis of the strain. We have chosen the MazF toxin protein to guide apoptosis. The MazF toxin protein encoded by the mazF gene specifically cuts the ACA sequence of the free mRNA, thereby inhibiting protein synthesis and causing cell growth arrest.
To obtain a suitable density of trigger promoters, we developed a model of the competitive kinetics of two microorganisms under limited resource conditions based on microbial growth kinetics and fermentation kinetics (see more details about model at: ). With this model, we predicted the density at which the engineered bacteria could act as inhibitors for different Malassezia concentrations. At 250 CFU/mm2 of E. coli Nissle 1917 on the scalp, it inhibits the growth of Malassezia under most conditions. Further experiments and calculations show that the triggering OD600 of the engineered bacterium is 0.21, which means when the microbial growth reaches a density of OD600 = 0.21 corresponding to PQS, E. coli Nissle 1917 will initiate programmed death, thus preventing the expansion and contamination of the microbiota and ensuring biosafety.
Fig.1The mechanism diagram of Iron limit switch
References：
[1] Guan, L., Liu, Q., Li, C., & Zhang, Y. (2013). Development of a Fur-dependent and tightly regulated expression system in Escherichia coli for toxic protein synthesis. BMC biotechnology, 13, 25. https://doi.org/10.1186/1472-6750-13-25
[2] Rottinghaus, A. G., Ferreiro, A., Fishbein, S., Dantas, G., & Moon, T. S. (2022). Genetically stable CRISPR-based kill switches for engineered microbes. Nature communications, 13(1), 672. https://doi.org/10.1038/s41467-022-28163-5
[3]商洁琳.(2014).两种群有限资源比值竞争动力学模型(硕士学位论文,山东大学) https://kns.cnki.net/KCMS/detail/detail.aspx?dbname=CMFD201402&filename=1014307494.nh
Lab Safety
Prologue
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.
Safety 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
Basic laboratory rules
1. All laboratory personnel must receive safety education and training and pass the laboratory safety examination.
2. When entering the laboratory, each experimenter should wear personal protective equipment (PPE) including lab coat, goggles, gloves, mask, etc. PPE, such as contaminated laboratory clothes and gloves, should not be worn when leaving the laboratory.
3. It is forbidden to eat, drink and stay in the laboratory.
4. The laboratory has an emergency evacuation roadmap, and the escape channel is unimpeded. The laboratory is properly equipped with fire-fighting equipment, emergency sprayers, eye washers and medical first aid kits and is regularly maintained.
5, To ensure the safety of the use of electricity and water in the laboratory, it is not allowed to use electrical appliances in a dangerous way. When using electric autoclave pot and other dangerous instruments, it is necessary to strictly comply with the operating procedures and require someone to watch at all times. The experimenter must check the water and electric switch when leaving the laboratory.
6. Hazardous chemicals should comply with relevant national and school regulations. Laboratory personnel should understand the hazardous characteristics of the chemicals used, safety protection knowledge, storage methods, waste treatment, emergency treatment methods, etc.
7. Hazardous waste and general waste should be stored separately, and hazardous waste labels should be posted.
8. Before leaving the laboratory, the experimenter must keep the experimental table clean and tidy, and put all the used instruments and drugs back in their original positions.
Instrument safety
In the molecular laboratory, we use ultra-clean table, PCR instrument, electrophoresis apparatus, double steam engine, centrifuge, metal bath, water bath, bacterial shaker and nano-droplets etc. In the cell lab, we use biosafety cabinets, cell incubators and microscopes. A clear guide for safe use of equipment is posted next to all experimental equipment, and special personnel check various facilities in the laboratory every day to eliminate possible risks. Before operating the equipment, we must read the operating instructions and accept the teacher's instruction. When operating the equipment, we must strictly follow the instructions, always close to the equipment during use, and promptly turn it off after use.
Other safety details
For some other important details, we also do very well.
Our laboratory is regularly sterilized with 75% ethanol, and the experimental table is clean and tidy, with reasonable layout, neat items and no crowding. Shelves placed neatly, no debris stacked. For waste recycling, we will use disinfectant to kill the remaining bacteria at the end of the experiment, and then pour it down the drain to ensure its sterility. For the waste of the molecular laboratory, we put it into the hazardous waste garbage bag and give it to the professional company for regular treatment. For cell lab waste, we put it in a closed container and then give it to a professional company for disposal.
