Team:Duke/Safety

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Safety in iGEM

Our project is a dCas9 powered gene circuit that induces a gene only in the presence of a known DNA sequence. However, we have chosen to showcase this construct in the context of a pressing problem in medicine: antibiotic resistance. We envision a system where the introduction neutral stimulus could produce a negative pressure on bacteria with antibiotic resistance via activation of a programmable cell death gene. The process could move existing bacterial populations away from antibiotic resistance when not using antibiotics.

We hope to perform a proof of concept experiment to test whether the presence of a plasmid can activate a reporter gene. We intend to mark initial trials with fluorescent proteins in order to easily isolate the effect of the circuit on transcription but then to extend the effect to the study of population growth patterns.

Safety Considerations of a BioSafety Level 2 Lab

Our research was conducted in a Biosafety Level 2 Lab and so standard protocol was followed for this level as per Duke policies. A link to policy overviews can be found here.

Our lab faces problems inherent in all molecular and synthetic biology research. E. coli, despite the docile strain, may still have adverse health effects if in the body. Thus, gloves are used in all lab procedures and hands are washed when leaving lab. Open food is not allowed in the lab. Ethanol is used to clean the lab bench of any bacterial contamination, in order to limit the spread of bacteria and plasmids produced for experiments.

Whenever potentially noxious chemicals are handled, they are done so in a chemical fume hood erring on the side of caution and gloves are changed immediately after handling potentially harmful chemicals such as Ethidium Bromide. Additionally, direct UV light exposure is limited when imaging and cutting gels by use of protective glasses and UV reflective coat as well as by limiting the amount of body exposed to the UV lamp while cutting the gels.

The project specifically deals with genes that trigger cell death and lysis, and as such we have attempted to seek out safety information and explore safer alternative genes. Although none of these genes are dangerous to human cells when produced at the level needed in lab, constructs are designed to only be expressed during experiments. As mentioned, among the novel cell death genes are alternatives to existing BioBricks shown to be less hemolytic to humans.

Any long term applications in microbial populations within humans should place safety as the primary concern. As such, the cell death gene would need to be with minimized side effects on the patient. Further, the long term successful implementation would work on a largely population level, so the spread of the antibiotic detecting plasmid would be important to the overall phenotypic change away from antibiotic resistance. But particular care would be necessary to limit unwanted infection of synthetic plasmids into those who do not want the plasmid. Care should also be taken to avoid any pathogenicity that could arise from the added plasmid and cell death gene.