Team:StanfordBrownSpelman/Amberless Hell Cell

Amberless Hell Cell

For an application of synthetic biology where live, genetically-modified cells will come in direct contact with the environment, such as biological sensors on 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 solve both of these issues, and therefore to create an ideal chassis for synthetic biology in environmental applications, we will combine two research goals:

1. The "Hell Cell" project by the 2012 Stanford-Brown iGEM team isolated genes from extremophile bacterial species and inserted them into Escherichia coli, in order to create bacteria that are resistant to extremes in pH, temperature, and moisture. We sought to further characterize, improve, and search for new resistance genes that would help our chassis survive in earth and space applications.

2. The Church Lab at Harvard Medical School in 2013 created a strain of E. coli (C321.ΔA) in which all 321 instances of the UAG ("Amber") stop codon in the E. coli genome had been replaced with the UAA stop codon [1]. Release factor 1, which terminates translation at UAG, was also removed. With this system, the Church group incorporated artificial amino acids with a tRNA that recognizes UAG as its codon.

We developed a novel approach for preventing 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 [2]. 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.

Approach & Methods

Methods here.

More methods here.

More methods here.

Click here to download our project journal, which details our design and engineering process and included descriptions of the protocols we developed and used.

Results

Results go here.

References

1. Lajoie MJ et al. (2013) Genomically Recoded Organisms Impart New Biological Functions. Science 342: 357-60. PMID: 24136966.

2. Thorbjarnardóttir, S. et al. (1985) Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene. J. Bacteriol. 161: 219–22. PMID: 2981802.