Saccharomyces cerevisiae, or baker's yeast, is the chassis we have chosen for our project. Yeast is commonly used in brewing and baking and poses a low risk of infection under most circumstances. Infections are treatable and non-lethal. According to the World Health Organization laboratory biosafety manual, Saccharomyces cerevisiae is a risk group 1 organism with low risk to individuals or the community of transmitting disease. Many yeast strains we work with in the lab lack multiple essential biosynthetic pathways, including those for leucine, histidine, and uracil. Therefore, not only do our strains depend on media supplementation of these components in order to survive, they are also less fit than their wild type counterparts. If released into the wild, our lab yeast strains would fare poorly and we do not anticipate any concerns to public or environmental safety.
Other chemicals, reagents, and equipment used in the lab that pose a safety hazard are ethidium bromide (EtBr) and ultraviolet (UV) light, which are used to visualize DNA on agarose gels. Exposure can be minimized by wearing lab coats, nitrile gloves, closed-toe shoes, and taking proper safety precautions such as isolating EtBr-exposed equipment to a single room. In the lab we use non-pathogenic E. coli strains for bacterial transformations. Although we introduce plasmids conferring antibiotic resistance (e.g. ampicillin and kanamycin) into these cells, in the absence of selection the plasmid should be readily lost and not pose a problem.
Because of the low risk, our research is conducted in a BSL1 laboratory. Each member is given personal protective equipment such as lab coats, gloves, and safety glasses and has undergone safety training both in the lab and with the Universities' laboratory safety education initiative. Equipment is sterilized in the autoclave before and after use and all waste is disposed of in biohazard bags that are incinerated at an onsite facility. Yeast and bacterial cultures are all treated with 10% bleach prior to disposal.
The majority of new BioBricks we are submitting are amplified and subcloned from yeast genomic DNA, including inducible yeast promoters and a series of yeast terminators. We have also submitted a part containing CYP2E1, a human cytochrome P450 gene whose encoded enzyme is responsible for converting ethanol into acetaldehyde. Using these parts, we have generated functional 'compound parts' to be expressed in yeast that should result in increased production of acetaldehyde at the expense of ethanol molecules. Acetaldehyde is a common compound found in nature and is produced on a large scale industrially. Although acetaldehyde (MSDS) is less toxic than ethanol, prolonged exposure to acetaldehyde is an irritant and possibly carcinogenic. However, the quantity of acetaldehyde produced and the scale of our experiments is far below these limits.
Our research follows the rules set by the Johns Hopkins Biosafety Office and the Johns Hopkins Medicine Institutional Review Boards. These guidelines can be found online on the Johns Hopkins Biosafety website. Our project has been registered with the office under experiments that use recombinant DNA. Following the NIH guidelines for research involving recombinant DNA molecules, the Saccharomyces cerevisiae chassis is exempt from institutional biosafety committee and does not require IRB approval. We have reviewed the new NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acids Molecules, effective March 2013, and our project still follows all exemption guidelines.
The goal of the light control project is to arrest the yeast cell cycle by exposure to a specific wavelength of light. The wavelength we are using, ~450nm, is in the visible light spectrum and exposure is not hazardous. If the project is successful, it may present a new safety switch, as the growth of cells could be reduced or even arrested due to exposure to ambient light, thus rendering our cells less fit beyond the context of a light-restricted environment. This is a variant of the 'Gene Guard' or 'Kill Switch', previously introduced to iGEM by teams such as Imperial College London who developed the toxin/anti-toxin in their bacterial strains (Auxin Project 2011).
"
