All of our team members were given a thorough safety training prior to the start of the iGEM program. A list of training programs that we attended can be found here http://rac.berkeley.edu/training.html. UC Berkeley EH&S office (http://www.ehs.berkeley.edu/) closely supervises research practices on campus, and our project was first approved by this office before its commencement.
Safety forms were approved on 9/18/13 by David Lloyd and Julie McNamara.
Our team worked only with the Escherichia coli and Saccharomyces cerevisiae species. E. coli K-12 is not considered a human or animal pathogen nor is it toxicogenic. Any concerns for E. coli K-12 in terms of health considerations are mitigated by its poor ability to colonize the colon and establish infections (http://epa.gov/oppt/biotech/pubs/fra/fra004.htm). S. cerevisiae is a GRAS organism (Generally recognized as Safe). Saccharomyces, as a genus, present low risk to human health or the environment. Criteria used to differentiate between species are based on their ability to utilize specific carbohydrates without relevance to pathogenicity (http://www.epa.gov/biotech_rule/pubs/fra/fra002.htm). Preventive measures such as sterilization of materials prior to disposal are in place to avoid any unforeseen risks.
We do not envision any of the parts we are using to be a safety concern in the lab. All of the parts we have cloned and expressed as well as their corresponding substartes and products have been documented previously by other authors. FMO's product, indigo, is known to be a low toxicity compound (http://www.ncbi.nlm.nih.gov/pubmed/3598883) and many of the safety practices surrounding use of indigo have been well documented in the literature.
In our envisioned dyeing process, indican would be used as a dyeing material. Once indican permeates fabric, our B-glucosidase would be added either as cell culture or purified enzyme to the jeans, which would then turn indican into indigo. Once the jeans have been sufficiently dyed, we would aim to: remove or eliminate bacteria from the cloth, remove or recycle unbound indigo, and remove or recycle unreacted indican. Indigo and indican are generally safe, though we would want to avoid large-scale environmental release. After dyeing fabric, several rounds of washes in detergent will likely eliminate any remaining bacteria. Our process, if scaled up to industrial levels, would have to undergo rigorous testing to confirm that the dyed product is cell-free. Because indican is soluble, it will wash off easily in detergent and water, and unbound indigo will likewise be removed.
After dyeing our cotton bears with indican and our purified B-glucosidase, we washed with 5% SDS (a detergent) several times and then autoclaved the cotton material before washing again with 1% detergent water to be sure that we have completely eliminated all biological activity before removing them from the lab.
Scaling our process would present the added safety concerns of building and maintaining a bioreactor facility. Any facility meant to grow indican-producing and B-glucosidase producing bacteria as outlined on our Human Practices page would follow standard bioreactor design regulations as well as waste and operation regulations.