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Team:Austin Texas/kit
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In order to re-code UAG, a synthetase/tRNA pair much be modified to effectively grab onto an ncAA. Various methods of directed evolution are typically used to modify a synthase such that it can grab onto and charge a non-canonical. The library of ncAA synthetases available have a ranging levels of reported efficiency and are not well characterized. The This year the UT iGEM Team created a test kit designed to characterize the efficiency of an ncAA synthetase/tRNA system. | In order to re-code UAG, a synthetase/tRNA pair much be modified to effectively grab onto an ncAA. Various methods of directed evolution are typically used to modify a synthase such that it can grab onto and charge a non-canonical. The library of ncAA synthetases available have a ranging levels of reported efficiency and are not well characterized. The This year the UT iGEM Team created a test kit designed to characterize the efficiency of an ncAA synthetase/tRNA system. | ||
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| + | [[File:NcAA kit incorpotation 10-11-14.png|300px|thumb|left]] | ||
The kit consists of a three plasmid system: pBLG, pFRYC, and pFRY. pBLG contains a gentamicin resistance as well as the ncAA synthetase/tRNA pair. pFRYC is the control plasmid and contains a kanamycin resistance gene and an IPTG induced reporter system. The reporter system is composed of sfGFP connected via a linker sequence upstream of mCherry RFP. pFRY is the experimental plasmid and is similar to pFRYC however the pFRY linker sequence contains an amber stop codon whereas the pFRYC linker does not. In a cell containing pFRYC, the ribosome will translate the sfGFP reporter, linker, and finally mCherry producing a fluorescent reporter of green and red resulting in a yellow fluorescent. In a cell containing pFRY, the ribosome will translate the sfGFP and terminate at the amber stop codon on the linker producing a green fluorescence. When pBLG and pFRY are present in the cell, the ribosome will incorporate an ncAA at the amber codon in the linker and continue translation producing both sfGFP and mCherry reporters if the synthetase/tRNA pair encoded on pBLG effectively incorporate the ncAA. | The kit consists of a three plasmid system: pBLG, pFRYC, and pFRY. pBLG contains a gentamicin resistance as well as the ncAA synthetase/tRNA pair. pFRYC is the control plasmid and contains a kanamycin resistance gene and an IPTG induced reporter system. The reporter system is composed of sfGFP connected via a linker sequence upstream of mCherry RFP. pFRY is the experimental plasmid and is similar to pFRYC however the pFRY linker sequence contains an amber stop codon whereas the pFRYC linker does not. In a cell containing pFRYC, the ribosome will translate the sfGFP reporter, linker, and finally mCherry producing a fluorescent reporter of green and red resulting in a yellow fluorescent. In a cell containing pFRY, the ribosome will translate the sfGFP and terminate at the amber stop codon on the linker producing a green fluorescence. When pBLG and pFRY are present in the cell, the ribosome will incorporate an ncAA at the amber codon in the linker and continue translation producing both sfGFP and mCherry reporters if the synthetase/tRNA pair encoded on pBLG effectively incorporate the ncAA. | ||
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NEXT: Create experimental section, data section, discussion/conclusions section,. | NEXT: Create experimental section, data section, discussion/conclusions section,. | ||
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==ncAA Table== | ==ncAA Table== | ||
Revision as of 21:29, 11 October 2014
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