Choosing a non-pathogenic chassis

Bacterial strain: Escherichia coli K-12

Escherichia coli K-12 is not a threat to humans, animals (chickens, pigs, and calves), plants or the environment, according to the 1997 Final Risk Assessment of Environmental Protection Agency (EPA). “Any concerns in terms of health considerations are mitigated by its poor ability to disseminate, colonize the colon and establish infections in a murine model” (Smith et al., 2010). In addition, the chance of the insertion gene mutating the bacterial strain to be hazardous is minimal as the inserting genetic materials have to meet the EPA’s criteria: limited in size, well characterized, free of certain nucleotide sequences, and poor mobility.

Choosing parts that will not harm humans/ animals/ plants

Inserts:

Our GEOs are composed of standard promoters, ribosomal binding sites, terminators and fluorescent reporters, and have no known risks. The function and known homologs are classified by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and researched (mainly via PubMed). For more information on the researcher, public, and environmental safety refer to Safety Q & A

It is illegal and immoral to work with a system if it could be substituted for a safer suitable system that is also compatible with the intended work even if the alternative is more difficult or expensive to acquire. In our project, safety is favored before monetary issues. Therefore an attempt is always made to locate a better host, donor, vector, insert, or system for humans, animals and the environment in order to ensure that the system is the safest it can be.

Since Ethidium bromide is a very toxic agent and acts as a mutagen (intercalates double stranded DNA), we have changed our nucleic acid staining molecules to Invitrogen SYBR Safe, which is significantly less toxic and better for the environment (may have direct disposal into the wastewater drainage systems). In case EtBr or Invitrogen SYBR Safe affects DNA biological processes, these substances are added in minimal concentration to the agar solution before it solidifies.

We usually use UV radiation to visualize the stained DNA in agar gels. However, long exposure to UV light is harmful, carcinogenic, and destroys vitamin A. Therefore, we replaced UV transilluminator with digital gel imaging systems to avoid close UV contact. This reduces the necessity of special safety lenses and other special equipment that were used to block the harmful effects of ultraviolet. UV is also used in laminar flow cabinets for sterilization and decontamination purposes. For safety precautions, we have limited the sterilization process to 30 minutes with no other personnel in the laboratory.

At first paraformaldehyde was considered a good choice because the substance was known for its fixation properties. However, the level of toxicity led many to question our disposal system. In addition, paraformaldehyde did not fulfill our project needs. We wished to have constant fluorescent intensity with decreasing or constant cell count but the use of paraformaldehyde hindered this ability as it lacked constant fluorescent intensity.

Next we considered sodium azide. This substance is a strong respiratory toxin which inhibits the activity of cytochrome oxidase and is similar to a bacteriostatic agent. However, this chemical is also highly toxic to mammals. Therefore, we did not experiment with sodium azide.

Sodium azide led us to explore all kinds of bacteriostatic agents. Tetracycline, ampicillin, and sulfonamide are just some of the bacteriostatic agents that we researched. Tetracycline attaches to 30s ribosomal subunit and inhibits protein synthesis causing bacteria cell count to slowly decrease. Ampicillin inhibits additional formation of cell membrane and sulfonamide obstructs DNA replication, therefore in both cases cell splitting becomes impossible.

Safety is one of the most important aspects of iGEM. When biosafety is not considered properly, many life forms can be destroyed. To start, we decided to experiment with paraformaldehyde. The renowned fixation properties led us to believe that our E.coli can be considered nonliving while it continues to function. However, the results of our experiments were not favorable. In addition, the toxicity of paraformaldehyde itself raised questions. The search for other alternatives led us to bacteriostatic agents.

There are numerous varieties of bacteriostatic agents, such as glycylcycline, macrolide, oxazolidinone, and tetracycline. In the preceding experiments, tetracycline, ampicillin, and sulfonamide were researched. These substances represent different types of inhibition to stop cell replication. Tetracycline attaches to 30s ribosomal subunit and inhibits protein synthesis causing bacteria cell count to slowly decrease. Ampicillin inhibits additional formation of cell membrane and sulfonamide obstructs DNA replication. Therefore, in both cases cell splitting becomes impossible.