IGEM:Paris Bettencourt 2012/Background

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Recent advances in the field of synthetic biology have created an ever increasing desire to apply genetically modified bacteria (GMB) in an ecological setting. Synthetic biologists aspire to design possible applications of synthetic biology: bioremediation, agriculture, resource processing, biofuels, etc. These projects, while interesting often as commercial or humanitarian ventures, so far fail to describe how a genetically modified micro-organism could be safely and effectively released into the environment. We seek to make steps toward enabling the use of large quantities of genetically engineered bacteria the environment by addressing the safety and practicality concerns.

The first genetically modified microorganism ever to be use in the environment was the “Ice-minus bacteria”, a strain of P. syringae that lacked functional Ice nucleation-active protein. Normally these bacteria have a surface protein that facilitates the nucleation of ice crystals. A simple mutation disables the ability for water to freeze efficiently on the surface of these bacteria, and it occurred to engineers that they could be used to protect crops from damage during frosts. In 1987 it was sprayed on a strawberry field in California before a frost, and although results were promising, it was also suspected that activists destroyed some of the plants in protest.

The fear is that dangerous recombinant genes could upset balance in the environment. Some claimed, for instance, that the ice-minus bacteria could upset normal rainfall because the Ice nucleation-active protein may be involved in normal cloud formation. This may be an alarmist viewpoint, but for each individual application of genetically modified bacteria there will be a host of such safety concerns. Most, since the events 1987, have shied away from the notion of environmental applications of genetically modified bacteria. We believe, however, that synthetic biology has such potential as a tool for use in the environment that we can no longer ignore it. It is time to open discussion of how we can begin to use the environmental GMB safely and ethically.

Genetically engineered microorganisms can perform tasks that are more complex than chemical agents. The secret, of course, lies in their protein enzymes that can perform various forms of metabolism at great specificity and at normal temperatures. More than just introducing a chemical catalyst into the environment, bacteria can bring reactants through complex metabolic pathways such as the nitrogen fixation performed by the Rhizobium bacteria – a starting point for a potential replacement of the costly Haber process being worked on by the US Agricultural Research Service. The majority of current research interest in environmental GMB is for the purpose bioremediation – the in situ processing and neutralization of pollutants. iGEM teams often focus on this subject, such as the 2011 Caltech team that created a strain of E. coli to degrade endocrine-disrupting chemicals such as estrogen in bodies of water. Academic research has had a particular interest in the bioremediation of heavy metals in the environment.

How can GMB be used in the environment? How can they adhere to NIH standards and follow guidelines set by the US Presidential Commission for the Study of Bioethical Issues? We believe the answer is that experimental standards must be created to quantify the containment of recombinant DNA. Every GMB created will have its own unique genetic characteristics. We intend to create a tool that can be used by other iGEM teams, academic researchers, companies, to measure their own rate of gene transfer. This will be a unifying standard – and perhaps inspire the creation of a more perfected one – to validate the use of any particular GMB in the environment.

The precedent in the United States for measuring safety of genetically modified crops is based upon a concept called “substantial equivalence,” in which biochemical profiles of GM crops are compared to natural types. If sufficiently identical in their profiles, the GM crop and natural types are deemed the same food. The concept fails to acknowledge the eventuality of the transfer of recombinant genes to other species and regions. We want to focus on gene containment by creating a delayed action gene restriction system that will greatly – hopefully completely – destroy any recombinant DNA existing in our modified strains. A cell death system is also being considered.

The largest technical challenge facing synthetic biologists in applying GMB in the environment is the unpredictability of genetic and physiological behavior of bacteria. Environments such as soil and ocean expose bacteria to a gamut of unknown chemicals and heterogeneous conditions. Almost never will bacteria behave in the same way as they do in the laboratory when simply sprayed on terrain. For this reason and others, we will create beads – permeable micro-environments – for our bacteria. Probably made from alginate or similar medium, the beads will aid in containing genes by holding bacteria stationary. They will offer a more homogeneous and predictable environment so that we can be more confident in claiming that our system will work as intended. The beads will also help ensure a degree of survival for our bacteria. The homeostasis established in natural environments is very hostile to GMB, for instance due to predatory protozoa.

Our research will be performed with close reference to a paper by Torres et. al A dual lethal system to enhance containment of recombinant microorganisms. They created a dual toxin-antitoxin system that demonstrated greatly reduced transfer of recombinant genes. They measured gene transfer of the two systems both individually and when functioning together in the same organism. Resulting gene containment was not a best-case scenario predicted by multiplying the individual rates of gene transfer, there was still an improvement.

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