For any application of biological engineering where live, genetically-modified cells will come in direct contact with the environment, for instance as components of a UAV. Two concerns must be addressed: first, the cells need to be resistant to widely-varying conditions that may be present in the environment; second, in order to address ethical concerns about releasing genetically-modified organisms, it is desirable to reduce horizontal gene transfer from the engineered cells into cells naturally present in the environment. In order to face both of these issues, and therefore to create an ideal starting strain for genetic engineering for environmental applications, we will combine two previous projects:
1. The "Hell Cell" project by the 2012 Stanford-Brown iGEM team isolated genes from extremophile bacterial species and inserted them into E. coli, in order to create E. coli that are resistant to extremes in pH, temperature, and moisture.
2. A lab at Harvard Medical School in 2013 created a strain of Escheria coli in which all UAG ("Amber") stop codons in the entire genome had been replaced by other stop codons. Release factors that recognize UAG as stop were also removed. This allows for the insertion of artificial tRNA's that translate UAG into an amino acid.
We plan to use this approach to prevent horizontal transfer of engineered genes into the environment by inserting a UAG-leucine tRNA, and using UAG for leucine in all of the inserted, engineered genes. Because these genes will not be read correctly in other organisms (the UAG will be read as stop, so proteins will be truncated), the engineered genes will not have any effect in naturally-occurring bacteria in the environment. Our project will involve synthesizing UAG-leucine coded versions of the Hell Cell genes and inserting them into the amberless E. coli strain, along with a UAG-leucine tRNA. This will create a strain of bacteria that is both resilient and safe for environmental applications, for example as a biosensor added to the BCOAc membrane using the biotin/streptavidin interaction mentioned above.
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Results
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Methods & Safety
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