Difference between revisions of "Part:BBa K2201343"
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This part is an improved version of the pFRY plasmid ([https://parts.igem.org/Part:BBa_K1416004 BBa_K1416004]) and pRXG ([https://parts.igem.org/Part:BBa_K2020040 BBa_K2020040]) consisting of a mRFP domain which is connected by a linker sequence containing an amber stop codon with a sfGFP domain. The expression of the plasmid results either in red fluorescence, or - if the ncAA is incorporated at the amber stop codon within the linker site - in both: red and green fluorescence. By comparison of fluorescence levels it is possible to determine incorporation efficiency of the generated synthetase variants.
 
This part is an improved version of the pFRY plasmid ([https://parts.igem.org/Part:BBa_K1416004 BBa_K1416004]) and pRXG ([https://parts.igem.org/Part:BBa_K2020040 BBa_K2020040]) consisting of a mRFP domain which is connected by a linker sequence containing an amber stop codon with a sfGFP domain. The expression of the plasmid results either in red fluorescence, or - if the ncAA is incorporated at the amber stop codon within the linker site - in both: red and green fluorescence. By comparison of fluorescence levels it is possible to determine incorporation efficiency of the generated synthetase variants.
  
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[http://2017.igem.org/Team:Bielefeld-CeBiTec/Improve We] had a little trouble in using it at our own synthetases. We investigated that there were some issues with the choice of RFP and GFP for the system and decided to improve the part by using CFP and YFP, for they form a FRET system which leads to a more accurate distinction between the partial (CFP) and the whole (CFP-YFP) expressed fusion protein.
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[http://2017.igem.org/Team:Bielefeld-CeBiTec/Improve Our team] had a little trouble in using it at our own synthetases. We investigated that there were some issues with the choice of RFP and GFP for the system and decided to improve the part by using CFP and YFP, for they form a FRET system which leads to a more accurate distinction between the partial (CFP) and the whole (CFP-YFP) expressed fusion protein.
  
 
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Revision as of 20:09, 24 October 2017


Fusion protein of CFP and YFP with an amber codon in the linker under T7-promotor control

Introduction

This part is an improved version of the pFRY plasmid (BBa_K1416004) and pRXG (BBa_K2020040) consisting of a mRFP domain which is connected by a linker sequence containing an amber stop codon with a sfGFP domain. The expression of the plasmid results either in red fluorescence, or - if the ncAA is incorporated at the amber stop codon within the linker site - in both: red and green fluorescence. By comparison of fluorescence levels it is possible to determine incorporation efficiency of the generated synthetase variants.

[http://2017.igem.org/Team:Bielefeld-CeBiTec/Improve Our team] had a little trouble in using it at our own synthetases. We investigated that there were some issues with the choice of RFP and GFP for the system and decided to improve the part by using CFP and YFP, for they form a FRET system which leads to a more accurate distinction between the partial (CFP) and the whole (CFP-YFP) expressed fusion protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 770
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1360
    Illegal AgeI site found at 1472
  • 1000
    COMPATIBLE WITH RFC[1000]