Difference between revisions of "Team:William and Mary/Measurement"

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For our project this year we successfully designed and characterized an accessible and modular degradation based system for the control of gene expression speed. Utilizing an <i>E. coli</i> orthogonal tmRNA degradation system consisting of a  <i>Mesoplasma florum</i> Lon (mf-Lon) protease [1] and highly engineered tmRNA tags [2] with a range of protease affinities, we were able to create a qualitative and quantitative gene expression speed change that was dependent on degradation rate. To do this we first had developed a time course measurement protocol that would allow robust and reproducible single cell measurements. Developing this method was time intensive, and meant that we spent a large portion of the summer without getting high-quality gene expression speed data, but ultimately our final <a href='https://2017.igem.org/Team:William_and_Mary/Protocols' style='text-decoration: underline;'>time course protocol</a> ensured that we got robust, reproducible data that we could feel confident in.
 
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<div style = 'padding-left: 190px; padding-bottom: 10px;font-size: 25px' ><b>References</b></div>
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[1] Eyal Gur and Robert T Sauer. Evolution of the ssra degradation tag in mycoplasma: specificity switch to a different protease. Proceedings of the National Academy of Sciences, 105(42):16113– 16118, 2008.
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[2] D Ewen Cameron and James J Collins. Tunable protein degradation in bacteria. Nature biotechnology, 32(12):1276–1281, 20
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Revision as of 19:58, 1 November 2017



Overview
For our project this year we successfully designed and characterized an accessible and modular degradation based system for the control of gene expression speed. Utilizing an E. coli orthogonal tmRNA degradation system consisting of a Mesoplasma florum Lon (mf-Lon) protease [1] and highly engineered tmRNA tags [2] with a range of protease affinities, we were able to create a qualitative and quantitative gene expression speed change that was dependent on degradation rate. To do this we first had developed a time course measurement protocol that would allow robust and reproducible single cell measurements. Developing this method was time intensive, and meant that we spent a large portion of the summer without getting high-quality gene expression speed data, but ultimately our final time course protocol ensured that we got robust, reproducible data that we could feel confident in.
References
[1] Eyal Gur and Robert T Sauer. Evolution of the ssra degradation tag in mycoplasma: specificity switch to a different protease. Proceedings of the National Academy of Sciences, 105(42):16113– 16118, 2008.
[2] D Ewen Cameron and James J Collins. Tunable protein degradation in bacteria. Nature biotechnology, 32(12):1276–1281, 20