Listeria monocytogenes (hlyA gene)

The hlyA gene codes for Listeriolysin O. When human cells have engulfed Listeria cells by phagocytosis, Listeriolysin O becomes active and breaks open the phagosome, allowing the bacteria to live in the cytoplasm. Bacteria transformed with Listeriolysin O are invasive microorganisms with the ability to hide from the immune system. With Listeriolysin, bacteria can invade macrophages and then break open the phagosome, and the bacteria are free to live inside the cytoplasm. Genetically engineered bacteria that express both listeriolysin and invasin are able to invade any beta-1-integrin expressing cell type, and they can penetrate to the cytoplasm at high efficiencies.

The hlyA gene (coding for Listerolysin O) will be part of the transkingdom RNAi plasmid (TRIP) that the team will use to determine how well transkingdom RNA interference will work on different types of cancer cells and different oncogenes. We aim to design the bacteria so that it only targets cancer cells.

Yersinia enterocolitica (Inv gene)

The inv gene codes for the invasin protein. The invasin protein on the surface of a Yersinia cell interacts with beta-1-integrin receptors on the surface of eukaryotic cells. This triggers a signal transduction pathway, leading to endocytosis of the whole bacterium. Bacteria transformed with invasin are invasive microorganisms with the ability to hide from the immune system. Genetically engineered bacteria that express both listeriolysin and invasin are able to invade any beta-1-integrin expressing cell type, and they can penetrate to the cytoplasm at high efficiencies.

The inv gene (coding for invasin) will be part of the transkingdom RNAi plasmid (TRIP) that the team will use to determine how well transkingdom RNA interference will work on different types of cancer cells and different oncogenes. We aim to design the bacteria so that it only targets cancer cells.

Precautions taken with hlyA and Inv genes

Incubators, PCR, Thermocycler Station

Tissue culture hood/Biosafety cabinet

Open bench

The host strain is auxotrophic nonpathegenic E. coli, the DapA- mutant, whose growth is inhibited in the absence of lysine. We wore full personal protective equipment and worked only in specific areas designated only for handling these specimen. We were under the supervision of researchers who are experienced and qualified in working with BSL2 organisms.

All team members received lab safety training prior to lab work. Research compliance and administration is coordinated through Columbia University's RASCAL web-based application and through Columbia University's Office of Research Compliance and Training. Each member of the 2017 Columbia University iGEM team has undergone extensive training and attained certification in the use of recombinant DNA, biological safety and bloodborne pathogen precautions, and lab safety, chemical hygiene, and hazardous waste management. More information and resources about specific guidelines for safety can be found on the Office of Research Compliance and Training website at http://www.columbia.edu/cu/compliance/index.html. At Columbia University, the Environmental Health and Safety Department is responsible for the safety of biology labs. The department follows the guidelines set by the National Institute of Health (NIH) and does so through the "application of materials substitution, engineering controls, administrative controls and the use of personal protective equipment." A link to their guidelines can be found here: http://www.ehs.columbia.edu/bs.html. Safety topics discussed include lab access and rules, biosafety levels, biosafety equipment, good microbial technique, disinfection and sterilzation, emergency procedures, transport rules, chemicals, fires, and electrical safety specific to the lab space. Training was provided by the University biosafety office and by our PI's and Instructors. Recommendations as well as descriptions of mandatory safety and health standards are contained in the Occupational Safety and Health Administration (OSHA) Laboratory Safety Guidelines found at https://www.osha.gov/Publications/laboratory/OSHA3404laboratory-safety-guidance.pdf.

Ethical Risks and Safety Considerations

There are safety considerations when engineering a non-pathogenic strain of bacteria to possess such virulence factors as invasin and listeriolysin from yersinia and listeria, respectively. By engineering otherwise attenuated bacteria to possess these virulence factors, the bacteria can become pathogenic. Containment precautions must be taken to ensure the virulence factors do not proliferate through horizontal gene transfer. Additionally, though our project is a proposed treatment for aberrant gene expression at the post-transcriptional level for conditions such as cancer, it could also be used to induce such disease states. For example, if the shRNA released from the invading bacteria was to interfere with a tumor-suppressor gene rather than an oncogene, cancer could be induced.