Team:StanfordBrownSpelman/Amberless Hell Cell

From 2014.igem.org

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   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:
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   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:
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<b>1.</b> The <a href="https://2012.igem.org/Team:Stanford-Brown/HellCell/Introduction" target="_blank">"Hell Cell" project</a> by the 2012 Stanford-Brown iGEM team isolated genes from extremophile bacterial species and inserted them into <i>Escherichia coli</i>, 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.  
<b>1.</b> The <a href="https://2012.igem.org/Team:Stanford-Brown/HellCell/Introduction" target="_blank">"Hell Cell" project</a> by the 2012 Stanford-Brown iGEM team isolated genes from extremophile bacterial species and inserted them into <i>Escherichia coli</i>, 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.  

Revision as of 08:21, 17 October 2014

Stanford–Brown–Spelman iGEM 2014 — Amberless Hell Cell



Figure 1. Figure caption here.

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
We were interested in two avenues of research. The first was test our hypothesis that the amberless chassis would enable us to create an orthogonal protein expression system that would not function properly in other bacteria. By replacing a 2-4 Leucines with TAG stops in a gene, an organism that does not express the supP tRNA, which translates UAG into Leucine, would produce a truncated product. We have named this novel system Codon Security. The second goal of the project was to apply Codon Security to the Hell Cell genes from our 2012 team in order to limit the horizontal transfer of resistance genes when using synthetic biology in the environment. Applying the Leucine->Stop modifications to the Hell Cell genes and transforming them into amberless cells, we could make the world's first Amberless Hell Cell.


Figure 2. We transformed DH5-alpha and amberless cells with test plasmids containing GFP or aeBlue reporter genes with stop codons and the supP tRNA in order to establish a proof-of-concept for Codon Security.
More methods here.


Figure 3. Figure caption here.

Results
Results go here.


Figure #. Figure caption here.
More results here.


Figure #. Figure caption here.
More results here.


Figure #. Figure caption here.
More results 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.
Additional Information
Read about how our submitted Amberless Hell Cell idea was used as a government regulatory case study on synthetic biology. We then began a conversation with Dr. Mark Segal at the EPA about the regulation and safety of the use of engineered bacteria in the environment.

Submitted biobricks: We submitted 9 biobricks for this sub-project. Six of these bricks include parts that can enable other teams to use the Amberless chassis as a system for more responsible synthetic biology.
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