HuGenesS and safety, a project overview
Genetically engineered organisms often experience a reduction in fitness that leads to genetic instability of the engineered functions. Organisms with small genomes and high mutation rates such as viruses often make use of gene overlap to stabilize their genes.Our project aims to produce biobricks that contain two overlapping genes in different reading frames, generating so-called “entangled genes”. Entangling a gene of interest with an essential gene is likely to constrain its evolution.
In this project we first generated biobricks in silico using a recently developed software. They contained two entangled genes encoding different types of proteins. Some entanglements were designed to encode both:
(1) fluorescent proteins and antibiotic resistance
(2) antibiotic resistance and luciferase, an enzyme that produces light
(3) luciferase and CcdB, a toxin that is lethal for bacteria that do not express the antitoxin.
The biggest safety risk associated with our project is that we might increase the genetic stability of new engineered functions when we entangle them with an essential gene in our chassis. Should our bacteria be accidentally released, our genetic construction might have more chances to spread to other bacteria by horizontal gene transfer as its acquisition might be beneficial. This is why we thought that CcdB could be an efficient tool to prevent the acquisition of the biobrick by other bacteria.
Link to : the Safety form
Experiments in the laboratory
We worked towards the cloning of six biobricks and succeeded in obtaining two plasmids. The first one encodes a small thermostable fluorescent protein stable _YFP_LOV. The second contained two entangled genes knt-gfp predicted to confer resistance to aminoglycoside antibiotics and render the bacteria fluorescent. We tested whether the generated biobrick knt-gfp twas functional. Fluorescence measurements were carried out by flow cytometry and fluorescence microscopy. Antibiotic resistance was observed on selective agar plates and gradient plates.
Choice of chassis and biobricks to minimize risks
Our chassis (Escherichia coli strain DH5alpha) is non-pathogenic for humans, animals or plants. These strains carry mutations that ensure a low fitness if they were accidently released into the environment (auxotrophy, impairment in some DNA repair mechanisms). Our biobricks encoded proteins are not harmful to humans, animals or plants: fluorescent proteins, antibiotic resistance, luciferase, a bacterial gyrase inhibitor.
Biological safety laws and regulations in France
Our team researched the biological safety laws and regulations in France. We worked under these guidelines. They are found at the following links:
- Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual Directive within the meaning of Article 16(1) of Directive 89/391/EEC)
Safety precautions and training
The lab space, kindly provided by the Institute for Integrative Biology of the Cell, is designated as a biosafety level 1 laboratory. Current health and safety procedures are in place to prevent accidents, including no eating or drinking in the lab, wearing lab coats, regular hand washing, wearing gloves when required, keeping corridors and lab space devoid of clutter, labeling and proper storage of harmful substances. In addition, wearing face covers was compulsory inside the building to prevent person-to-person spread of CoViD-19. Our biological waste was autoclaved.
Our safety training was provided:
(1) online through our institutional training program: an online course with final questionnaire to obtain a certificate of proper training (Neo). The course covered the following topics:
- Lab access and rules (including appropriate protective attire and the absence of food or drink)
- Responsible individuals (departmental biosafety officer)
- Differences between biosafety levels
- Biosafety equipment (such as biosafety cabinets)
- Good microbial technique (such as lab practices)
- Disinfection and sterilization
- Emergency procedures
- Physical biosecurity
- Personnel biosecurity
- Chemicals, fire and electrical safety
(2) in person, through our advisors that included several confirmed researchers (Dr. Philippe Bouloc, Pr. Bury-Moné, Dr. Ombeline Rossier) and three PhD students (Luis Ramirez-Chamorro, Laetitia Maroc and Téo Hébra). They have extended experience working with E. coli and the experimental procedures that we performed. Téo and Laetitia were our lab instructors during the summer.