Biosafety

1. Would any of your project ideas raise safety issues in terms of:

• researcher safety: cross-linking in post-production steps of printing may have safety concerns depending on the type of cross-linking method
• public safety: the project could potentially lead to handling of bacteria in the home/retail, which necessitates ymeeting public safety codes for genetically engineered organisms
• environmental safety: disposal of bacterial waste/byproducts/supplies may need specialized infrastructure

2. Do any of the new BioBrick™ parts (or devices) that you made this year raise any safety issues?

• No: No novel BioBrick™ parts were created, and all parts and devices were constructed using past submitted parts, which other teams have documented to be safe.

3. Is there a local biosafety group, committee, or review board at your institution?

• Yes: Caltech Safety Office (http://safety.caltech.edu/)
• Our project has been approved by Caltech's Safety Office after inspection of the lab space, project proposal, and materials list.

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?

• Sometimes BioBrick™ parts are created with the intent to suppress cell growth or kill cells altogether: in these cases, teams should take extreme care to optimize the gene sequences such that mutations cannot result in a gene product that proves harmful to human (or domestic) life.
• In order to keep control of potentially harmful genes inserted into cells, care should be taken to have regulatory schemes such that these genes cannot be transferred to other organisms upon contact in the wild.
  • One mechanism would be to make a novel BioBrick™ gene only usable by a lab strain of cells lacking certain restriction enzymes, such that when this gene is transferred to a cell of any other strain, the DNA gets cut up at strategic locations rendering the gene useless.
  • Alternatively, genes could be constructed such that they require several specific prerequisites to function properly, and that these prerequisites are not attainable under anything but lab conditions.

Probability:

1. Could there be an unplanned event or series of events involving your project, resulting in either death, injury, occupational illness, damage to equipment or property, or damage to the environment? How likely is that going to happen?

• An unplanned event occurring upon our printer could disable the printer, however there is no risk of death or injury at all.
• There is a very small risk of releasing the bacterial cells outside the device, which could, in a sensitive person, cause mild illness if ingested.
• Damage to equipment or property is unlikely, if a bacterial culture leaks there is a chance of mild flooding (depending on the size of the printer constructed).
• There is minimal risk of damage to the environment, as the non-natural chemicals used in our device are at extremely low concentration and our cells, even if leaked out to the environment, would be quickly out-competed and die out.

2. Does your project require the exposure or release of the engineered organism to people or the environment (e.g. as medicine, for bioremediation)

• There is no direct contact between the operator of the device and the bacteria, although the bacteria will be in relatively close proximity and not necessarily isolated from the operator.

Hazard:

1. Could your device, when working properly, represent a hazard to people or the environment?

• No: the cells themselves are killed in the formation of the product, and post-processing steps can include steps to sterilize the final product before it is released from the machine.

2. Is your engineered organism infectious? Does it produce a toxic product? Does it interfere with human physiology or the environment?

• No: The organism used is a weakened lab strain of E. coli and is not infectious, does not produce toxic products, and does not interfere with human physiology or the environment.
• In addition, the product material used in the design, PHA, is a well-characterized and naturally-occurring substance that is not specifically known to cause health problems.

3. What would happen if one or several bioparts change their function or stop working as intended (e.g. through mutation)? How would the whole device or system change its properties and w hat unintended effects would result thereof?

• If any one biological part changes its function for the worse, the biological device would fail (at least in that one cell). The gene circuit is set up such that one deleterious change to the circuit will disable it.
• In failure, the device would most likely cease to function altogether, although in a worst case scenario it could trigger the cells to produce plastic all the time, creating a mass of PHA biopolymer, which is still innately harmless.

4. What unintended effects could you foresee after your engineered organism is released to the environment?

• If the cells were released to the environment and somehow survived, they could possibly exchange genetic material with other cells in the environment. However this would not be a problem as the cells do not have genes coding for either dangerous products nor special regulatory mechanisms that could enable outside pathogenic cells to become more dangerous.

5. Try to think outside the box, what is the absolute worst case scenario for human health or the environment, that you could imagine?

• The absolute worst case that could happen with our biological machine is that the cells get leaked out and somehow become pathogenic, after picking up genes conferring pathogenicity from the environment.
• This risk is highly likely, and in the case that it happens it should be addressed as if it were any other pathogenic E. coli strain.