Team:Edinburgh/Safety

1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?

Researcher safety: Escherichia coli JM109 is a widely used host strain with disabling mutations which is incapable of colonizing the human intestine, its natural habitat. The same is true of other similar E. coli strains which may be used. Citrobacter freundii is a close relative of E. coli which is not normally associated with human disease in healthy subjects, and is ACDP1 (unlike wild strains of E. coli, which are ACDP2). (Note: like many bacteria capable of growing at human body temperature, some strains of C. freundii are capable of infecting compromised hosts under unusual circumstances). All the organisms (E. coli, disabled and wild type strains, Shewanella oneidensis MR-1 and Citrobacter freundii) we work with are ADCP1 organisms.

Genes encoding sugar uptake systems, respiratory proteins and their accessory proteins, and common reporter genes, are not expected to increase pathogenicity in any way. Genes encoding counterselection enzymes such as nitroreductase and dehalogenase are expected to be toxic to cells in the presence of counterselective agents.

Vectors used will be standard, widely used, non-transmissible cloning plasmids such as pSB1C3 (Registry of Standard Biological Parts). These encode resistance to antibiotics such as chloramphenicol. This is not expected to pose any risk to human health, nor should such resistance determinants be passed to other bacteria. Part of this project will include the development of standard cloning plasmids which do not include antibiotic resistance determinants, which will decrease this risk even further.

Public safety: Our bio-electric interface could be made public in future in order to enable communications between electronic devices and bacteria, but this would take place in a contained system so users would not actually come in contact with anything potentially hazardous.

Environmental safety: E. coli host strains are unable to colonize the intestines, their natural environment. C. freundii strains are close to wild type and may be able to proliferate in the environment, but are not expected to pose any hazard to animals or plants or to the environment in general. Inserted genes are not expected to increase the ability of the organisms to survive in the external environment, or to cause harm to the environment or any other organism.

Conclusion: Resulting genetically modified microorganisms should pose no greater risk to human health than the host strains.

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?

We are working on the non-antibiotic selectable and counter-selectable markers including Sucrose hydrolase, dhlA,dhlB and nitroreductase, and creating bioelectric interface with NapC, cymA, ccm, mtrcAB.

Sucrose hydrolase allows growth on sucrose as a sole carbon source which would expand possible growth conditions. However, sucrose degradation is not known to be a pathogenicity factor for infection of humans, and the resulting genetically modified organism would pose no greater risk to human health than the host strains.

mtrCAB allows the bacteria to grow in anaerobic conditions.

The rest of the genes do not present any safety issues. NapC and cymA are used in the electron transport of E.coli JM109 and Shewanella respectively as part of the nitrate reduction pathway. The ccm gene cluster is involved in cytochrome c maturation. mtrCAB is a cytochrome c electron export system. Nitroreductase is involved in reduction of nitrogen containing molecules.

3. Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?

Yes, we have a Health and Safety board within the University of Edinburgh School of Biological Sciences. We have submitted a risk assessment form called ‘Genetically modified organisms (contained use) regulations 2000 - RISK ASSESSMENT FORM FOR ACTIVITIES INVOLVING THE USE OF GENETICALLY MODIFIED MICRO-ORGANISMS AND EUKARYOTIC CELL AND TISSUE CULTURE SYSTEMS’ and this has been approved by the board.

4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

Citrobacter could be made less pathogenic by mutating out the cephalosporinase (beta lactamase) gene that confers it ampicillin resistance. Most gram negative bacteria have this gene so it may offer some advantage to the organisms other than just antibiotic resistance. This way, if such a mutant strain were to be released into the environment, it might be less able to survive than the wild-type and if it were to somehow cause an infection, it would be easier to treat as ampicillin could be an effective antibiotic.

On the topic of antibiotic resistance, we suggest that antibiotic-resistant markers be used less frequently and swapped for non-antibiotic selectable and counter-selectable markers. This way, there would be no danger of antibiotic resistant plasmids spreading into the organisms in the wild.

Microbial fuel cells often use redox-active small molecules as mediators that carry the electrons from the organisms in the fuel cell to the electrode. These mediators are often toxic and/or expensive, so developing a mediatorless fuel cell system could be advantageous for the researchers and to the environment in several ways.