Safety considerations for our Dr. Phage
Our Dr. Phage
In our project, we make use of the native biological characteristics of phage to detect bacteria and produce our signaling protein LuxR in a positive correlation with the number of bacteria for downstream circuit in cell-free system to quantify. Here we will evaluate the potential safety threats in our project and how we minimize those risks to the lowest.
The potential safety concerns of our engineered phage for humans and the ecological system should be negligible after our overall evaluation.
What we did to minimize the potential hazard in our design?
Our project can be divided into mainly two systems: engineered phage detection system and bistable quantitative signal output system. Both systems seek for the minimized the safety risk throughout the whole project. For the upstream detection part, we utilize engineered T4 which carried with LuxR protein gene in its essential gene can infect and lyse the pathogen naturally. As a result of deficiency in essential gene of T4, our engineered phage cannot generate progenies, which does not possess any competitive edge in either natural environment or in the lab. For the downstream quantification part, we choose cell-free gene expression system as our chassis which may not cause risks to human compared to the organism-based chassis.
Because we may put a large quantity of our phage in the detection kit, we should consider the concerns raised from the accidental leakage of our engineered T4 phage.
Concerns on human health:
The influences of phage particles to human health are insignificant. Bacteriophages exists everywhere in our whole ecosystem and they are a kind of virus that only infect bacteria cell (Kutter et al., 2004). Currently, the virulent phage-based biocontrol agents are widely accepted due to their Generally Recognised as Safe (GRAS) status in food safety from the U.S. Food and Drug Administration because they do not encode virulence factor to induce human immune reaction (Fieseler et al., 2011). In addition, owing to the host specificity of phage, the phage used in food pathogen detection or as biocontrol agents will not be harmful to human’s resident microflora (Endersen and Coffey, 2020). In conclusion, the use of phage may not cause health hazards to human.
Concerns on ecological system:
For concern for the ecology, we mainly focused on the interaction between natural host and our phage. The survival of phage in the environment depends on the exposure degree to sunlight, because phages are sensitive to UV light and their lytic ability can be negatively influenced by UV light (Luria and Latarjet, 1947, Verheust et al., 2010). Our T4 phages may infect limited range of E. coli as the wild-type T4 and lyse bacteria finally. Besides, our phage cannot generate the new phage particles, which prevents the further infection. Moreover, the assembly-deficient phage lack of ability to encapsulate the host gene into new virions and transfer into another bacteria, which may significantly reduce the possibility of gene transfer between bacteria (Verheust et al., 2010). Therefore, we considered our engineered phage would not do harm to the environment.
Phage engineering using CRISPR/Cas9
CRISPR (clustered regularly interspaced short palindromic repeat)–Cas (CRISPR-associated) as a remarkable defense system found in bacteria and archaea has been applied as a popular gene editing tool widely. Because our target gene to edit just exists in the T4 phage, no off-targeting
During the research reading, we knew CRISPR-Cas system might also contribute to rapid evolution of phage. The researchers used CRISPR-Cas9 to editing phage found that the escape of wild type ghmC-T4 phage infections from the cutting of CRISPR/Cas9 complex accumulates mutations at extraordinary rates (Tao et al.). This leaded us to think that what are the consequences of the evolution phage and what should we do.
Consequence & Measure
The repaid evolution of phage will make the phages insensitive to the cutting of CRISPR/Cas system, possessing a fitness advantage in the population. Furthermore, since all mutant phages are resistant to CRISPR-Cas, they could contribute to bacterial evolution via horizontal gene transfer and other mechanisms, which may finally facilitate the co-evolution of phage and bacteria and contribute to the abundance of diversity of phage and bacteria (Koskella and Brockhurst, 2014).
Since we only conducted our experiments in the lab and we were not sure if our CRISPR-escape phages contain certain mutations in their gene, we could only prevent the leakage or the pollution when performing the experiments in the laboratory. Thus, we followed rigorous standardized operations in every experiment. Our experiments related to phage all conducted in the biosafety cabinet. Because the T-even phage is sensitive to 70% ethanol, we sprayed lots of ethanol on our lab coats and all the experimental materials before and after each experiment (Yamashita et al., 2000). Moreover, we sealed plates and tubes containing bacteriophage with film every time. Furthermore, we would autoclave our experimental waste and trash, which can further wipe out the possible mutants to ensure no mutant phage survives in the environment.
For the lab safety, we mainly follow the Health and Safety Handbook from Department of Biological Sciences Xi’an Jiaotong-Liverpool University (XJTLU).
All our microorganisms used in the experiment belong to Biological Safety Level 1 which are harmless to human and we performed our experiments all in BSL-1 laboratory.
All our teammates participated in the experiment were received necessary safety training and pass safety quiz before entering the lab. The contents mainly include the equipment training, such as autoclave, biosafety cabinet and centrifuge, and the introduction of general laboratories guidelines etc.
Personal Protective Equipment (PPE)
Laboratory coats must be worn for all work carried out in laboratories and when not in use, hung on the hooks provided (do not mix laboratory coats with outdoor wear). Laboratory coats and gloves must not be worn in the canteen, toilets or outside the laboratory area.
Disposable gloves must be worn when handling any toxic, hazardous or infectious materials.
Masks and eye protection shall be worn whenever splashes, spray, droplets, or aerosols of blood or other potentially infectious materials may be generated and there is a potential for eye, nose, or mouth contamination.
When using an ultra-sonicator, ear protectors must be worn. Insulated gloves and a face visor must be worn when handling liquid nitrogen containers (e.g. inserting/removing samples, filling container with nitrogen).
All PPE shall be removed immediately upon leaving the work area or as soon as possible if overtly contaminated and placed in an appropriately designated area for decontamination or disposal.
Biological or infectious waste is waste that has pathogens or biologically active material present in sufficient concentration or quantity so that exposure of a susceptible host could result in disease. Especially, to prevent the failure in other kinds of experiments caused by phage pollution. The two most common waste treatment methods utilized in our experiment are steam sterilization and chemical disinfection.
Steam Sterilization (Autoclave): Steam sterilization utilizes pressurized steam at 121 to 132 °C to kill pathogenic organisms that are present in the infectious waste. Steam sterilization process does not destroy the waste. Instead, it renders it noninfectious. Properly sterilized waste can be disposed of in the regular trash after placing the autoclaved bag containing the waste in a regular black household garbage bag.
Chemical Disinfection: Aqueous or solid biohazard waste that does not contain hazardous materials can be disposed of through the sanitary sewer provided it is treated prior to doing so. In order for this waste to be disposed on in the proper manner, the following criteria must be met:
Disinfectants used must have been shown to be effective against the microorganisms present.
Disinfectants used must have been shown to be effective against the microorganisms present.
ENDERSEN, L. & COFFEY, A. 2020. The use of bacteriophages for food safety. Current Opinion in Food Science, 36, 1-8.
FIESELER, L., LOESSNER, M. J. & HAGENS, S. 2011. 6 - Bacteriophages and food safety. In: LACROIX, C. (ed.) Protective Cultures, Antimicrobial Metabolites and Bacteriophages for Food and Beverage Biopreservation. Woodhead Publishing.
KOSKELLA, B. & BROCKHURST, M. A. 2014. Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities. FEMS microbiology reviews, 38, 916-931.
KUTTER, E., SULAKVELIDZE, A., SUMMERS, W., GUTTMAN, B., RAYA, R., BRüSSOW, H., ECOLOGY, P. & CARLSON, K. 2004. Bacteriophages: Biology and Applications.
LURIA, S. E. & LATARJET, R. 1947. Ultraviolet Irradiation of Bacteriophage During Intracellular Growth. Journal of Bacteriology, 53, 149-163. TAO, P., WU, X. & RAO, V. Unexpected evolutionary benefit to phages imparted by bacterial CRISPR-Cas9. Science Advances, 4, eaar4134.
VERHEUST, C., PAUWELS, K., MAHILLON, J., HELINSKI, D. R. & HERMAN, P. 2010. Contained use of Bacteriophages: Risk Assessment and Biosafety Recommendations. Applied Biosafety, 15, 32-44.
YAMASHITA, M., MURAHASHI, H., TOMITA, T. & HIRATA, A. 2000. Effect of Alcohols on Escherichia coli Phages. Biocontrol Science, 5, 9-16.