Team:CU-Boulder/HP/Silver

• Human Practices •
• General Application •

We very much wanted to continue off the work we had laid out at iGEM last year, using our mutant and photo-openable EutS compartments. However, last year’s iGEM team did not really focus on what you could use our compartments for. This year, we started out early by asking ourselves what human problem we could solve using nano-scale isolation and sequestration that could open on demand.

Small molecule drug delivery remains the biggest field in the pharmaceutical industry today. Every year, billions of these drugs are synthesized and consumed by humans in almost every corner of the world, for far ranging purposes. But there are problems with small molecule delivery: non-specificity of interactions and the relatively high concentrations you sometimes need to use to generate the effects are a major cause of “side effects” in pharmaceuticals. We wondered if this was a problem we could solve if we used our nanocompartments to deliver drugs to only their intended receptors, opening there on demand. This would cut down on widespread side effects throughout the body.

While we understood that this was by no means a perfect solution to this problem, as cost, storage, and administration could prove effective barriers, it could also allow for new small molecule treatments for conditions that might have never worked otherwise, and so we decided to forge ahead with this idea in mind.

To that end, we developed our Luciferase and TNF-alpha fusion cargo proteins, and assays which could test our EutS derived compartments as a means of treating cells or binding other ligands. While we still aren’t sure that would be usable or practical in its current state, it would nevertheless serve as a proof of concept that could be iterated on for years to come, potentially revolutionizing small molecule development with new approaches never before possible.

• Other Considerations •

Other concerns also arise when we consider the use of our compartment in vivo. These two considerations are the toxicity of Azobenzene and the potential immune response to EutS protein cages. At the least, computational epitope mapping of EutS monomers seems to indicate that a strong immune response to our compartments would not be very likely, a major plus as a drug delivery tool.

While no trials have been conducted on the toxicity of the non-canonical amino acid AzoPhe, it has been reported that azobenzene in the body can be reduced into phenylenediamine and benzidine. Phenylenediamine is only a mild potential allergen (seen here), but Benzidine is confirmed as a known human carcinogen (human carcinogen). If we were to continue to explore the possibility of compartment based drug delivery, we would likely eventually need to discontinue our use of AzoPhe in favor of a photoisomerizable compound with less potential for toxicity, or to use a different method of compartment breakage. Nevertheless, we had AzoPhe and an incorporation system for it already, and decided that at least as a proof of concept it would be worth forging ahead.

• Outreach •

Boulder is celebrated as being a very environmentally friendly city. However, this quality is often coupled with the fear of the “unnatural,” specifically in regards to genetically modified organisms (GMOs). Recently, Boulder county commissioners voted yes on a plan to ban GMO corn and sugar beets from being farmed within county lines.

As an iGEM team we appreciate synthetic biology as being a rapidly developing field in which genetic modification is of vital importance. When we initially began our project, we were curious about the local trend of GMO intolerance in Boulder. We conducted a survey of students attending the University of Colorado Boulder, from a wide range of majors, to gauge how our project would impact society. The results of this survey may be able to provide some insight into the students living in Boulder and their opinions regarding GMOs.

We received a total of 138 respondents, all of them being students aged between 15-24 (100%). When asked if they were for or against GMOs, 70 people responded “for” (50.7%) and 13 people responded “against” (9.4%). When the respondents who were against the use GMOs were asked why, the two most common reasons were that GMOs are “unnatural” and “harmful to the environment.”

Through this survey, we wanted to develop an in-depth understanding of why GMOs make people uncomfortable. Out of the total respondents, 89 people said they would eat an apple that contains protein from another organism (64.5%). However, 36 of those people would not eat the apple if that foreign protein came from a fish rather than an orange or mint leaf (40.5%). Also, 97% of people who decided between a GM fruit that is pest resistant and a non-GM fruit that has been treated with pesticides voted for the GM fruit, regardless of whether they were for or against GMOs.

The results discussed above indicate that University of Colorado Boulder students do not reflect the local community’s general bias against GMOs. It is evident, however, that confusion regarding GMOs stems from the “unnatural” origins of transgenic material and pesticide use. Since 115 people did not think enough information is available about GMO foods (83%), there is an obvious deficit in GMO education. With this information we as a team can work to educate the public, clarify these misconceptions, and reduce stigma against GMOs.