The main goal of our project is to safely engineer microbial communities by selectively expressing gene of interest (GOI) in specific species, which today is mainly limited due to horizontal gene transfer (HGT) that allows propagation of the GOI within other members of the community [1,2]. HGT events lead to unintended and unanticipated consequences, particularly in the field of health and ecology. We aim to combat this limitation by developing a software capable of modifying gene components to be optimally expressed in the desired bacteria via an engineered plasmid, while deoptimize in the other. In other words, our technology might provide a solution for safe bioengineering of the microbial community even if HGT takes place.
Nevertheless, just because you have your seatbelt on doesn't mean you can drive like crazy. Our software, which functions like a seatbelt, strives to increase safety when applying biological engineering. However, in a case of poor optimization score due to close taxonomic classification of bacterial pairs in the community, additional degrees of safety will be provided by the software, in the form of induce-toxicity in deoptimized bacteria. In this mechanism, the plasmid will include a toxic gene under the regulation of an inducible promoter, that is activated by a protein or molecule existing in the deoptimized bacteria only. Then, the toxic gene will be expressed to kill the deoptimized bacteria that received the plasmid by HGT, while leaving the optimized bacteria intact (see Fig.1).
Moreover, in an effort to prevent software abuse and to increase biosecurity, a feature for the detection of known harmful genes has been added to our algorithm. This feature can recognize known genes related to toxin production, the spreading of infectious diseases, and antibiotic resistance. When sequences are inserted by the user for optimization purposes, the software can halt the analysis and block the user if it detects any pathogenic sequences. This reduces the possibility of misusing the software for bioterrorism, biological warfare, or intended ecological damage. You can read more about this in our Software and Ethics sections.
Figure 1:Induce toxicity in deoptimized bacteria.
HGT is responsible for engineered plasmid transmission into deoptimized bacteria. When the software’s algorithm predicts restricted optimization, a toxic gene under an inducible promoter is added to the plasmid. An activator known to be found in deoptimized bacteria only then activates the promoter that leads to the transcription of the toxic gene, which in turn kills the deoptimized bacteria.
- Vos M, Hesselman MC, Te Beek TA, van Passel MWJ, Eyre-Walker A. Rates of Lateral Gene Transfer in Prokaryotes: High but Why? Trends Microbiol. 2015 Oct;23(10):598-605.
- D. Sun, K. Jeannot, Y. Xiao, and C. W. Knapp, “Editorial: Horizontal gene transfer mediated bacterial antibiotic resistance,” Frontiers in Microbiology, vol. 10, 2019.
Before any lab work could be performed, every lab-working team member completed and passed Tel Aviv University’s BioSafety courseware.
The courseware covered the following Biosafety Topics:
In the biology lab, we worked with various dangerous materials, especially bacteria that can pose a risk if they penetrate the body. The damage can be expressed immediately or after many years. Bacteria might penetrate the body through:
- Airborne - breathing in aerosols that are being produced during lab activity.The microorganisms we work with do not pose a health risk and therefore creating aerosols is not a concern.
- Skin contact - penetration through the skin.We make sure to use gloves and a lab coat.
- Swallow - penetration to the digestive system can occur via contaminated hands or when drinking and eating in the lab.Entering food and drinks to the lab was strictly prohibited. We make sure to use gloves and wash hands at the beginning and the end of every process.
To date, Tel-Aviv University is allowed to work in biosafety levels of BSL1-2+.We worked in BSL1, meaning that the work only included using bio-materials that are not known to cause ailments in humans or damage the environment. We make sure to use designated bags for biological waste and to disinfect work areas at the beginning and the end of every working day, as well as in case of a spill.
Any work in the lab was done with personal protective equipment: lab-coat, gloves, long pants and closed shoes, and wearing goggles and face protection when needed.During safety guidance, we have trained how to manage spills of toxic or bio-contaminant substances and the immediate procedures in case of eye/body contact with various materials.Additionally, we have completed fire safety training and assured that all members are familiar with the locations of the fire extinguisher and the emergency exits of the building.
All safety instructions are available on the following website:Safety Instructions
Biological waste was collected in a designated biohazard bag and bin both marked as bio-hazard and sterilized in the autoclave for 20min.
We have frequently prepared DNA electrophoresis gel with ethidium bromide to stain the DNA. Since ethidium bromide is cytotoxic, it was handled with extra precaution. All gels were prepared by melting agarose within the TAE buffer in a designated microwave. Heat-protective gloves were used during the procedure, and the ethidium bromide was added in drops when the solution cooled down just before the solidification point. At the end of use, all gels were collected to a designated bin that was collected by the safety unit of the university.
Our work didn't include any pathogens.
A file of lab safety instructions and protocols were available in the lab, including the contact numbers of the security unit and emergency.
Safety experimental design
We chose to test our concept only on safe strains of bacteria, B. subtilis and E. Coli K12 , . We specifically chose strains unable to survive out of the lab, or cause any threat to the environment or lab personnel.
As an extra precaution, all bacteria was considered biohazard and handled as such.
For our POC experiment, we optimized and deoptimized the gene mCherry for each model organism. We chose this specific gene for two reasons:1. It is easy to follow the expression of this gene by fluorescent tests. 2. It is safe and does not pose a risk to the workers or to the environment.
We used vectors that we received from Avigdor Eldars's lab. One of them contained the mCherry gene that was later replaced by the newly synthesized gene, and the other one did not and served as control. Both of the vectors contain Chloramphenicol resistance gene as a selective marker.