Team:Austin Texas/kit

From 2014.igem.org

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[[File:NcAA kit incorpotation 10-11-14.png|300px|thumb|left|Figure 1]]
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[[File:NcAA kit incorpotation 10-11-14.png|400px|thumb|left|Figure 1 Schematic demonstrating the gene expression of the kit plasmids under different growth conditions.  Need to revise this figure.  The current text in the image, and how it is positioned is not ideal.]]
The kit consists of a three plasmid system: pBLG, pFRYC, and pFRY. pBLG contains the ncAA synthetase/tRNA pair to be tested as well as a gentamicin resistance gene. pFRYC is the control plasmid and contains the IPTG-induced reporter system and a kanamycin resistance gene. The reporter system is composed of RFP and sfGFP fused via a linker sequence between the two. pFRY is the experimental plasmid and is nearly identical to pFRYC with the exception that its linker sequence contains an amber stop codon in the middle of the linker whereas pFRYC contains a codon for tyrosine in the same location. In a cell containing pFRYC, the ribosome will translate the RFP reporter, linker, and finally sfGFP, producing red and green fluorescent proteins that result in visible yellow fluorescence. 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 RFP and sfGFP 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 the ncAA synthetase/tRNA pair to be tested as well as a gentamicin resistance gene. pFRYC is the control plasmid and contains the IPTG-induced reporter system and a kanamycin resistance gene. The reporter system is composed of RFP and sfGFP fused via a linker sequence between the two. pFRY is the experimental plasmid and is nearly identical to pFRYC with the exception that its linker sequence contains an amber stop codon in the middle of the linker whereas pFRYC contains a codon for tyrosine in the same location. In a cell containing pFRYC, the ribosome will translate the RFP reporter, linker, and finally sfGFP, producing red and green fluorescent proteins that result in visible yellow fluorescence. 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 RFP and sfGFP reporters if the synthetase/tRNA pair encoded on pBLG effectively incorporate the ncAA.  
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[[File:NcAA Kit Plasmid Alignment 10-11-14.png|500px|thumb|right|Figure 2]]
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[[File:NcAA Kit Plasmid Alignment 10-11-14.png|600px|thumb|right|Figure 2 Text needs to be bigger.  I can't read the names on the plasmid.  Make the font in the original image bigger, if possible.]]

Revision as of 14:58, 12 October 2014

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