Part:BBa_K2201343

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.

Properties

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Figure 1: Relative absorption and emission of CFP. The highest value equals one. The maximal absorption lays at ~430 nm (grey line). The emission maximum lays at ~475 nm (blue line).

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Figure 2: Relative emission spectrum (CFP signal: excited at 430 nm; YFP signal: excited at 475 nm) of the CFP-YFP system cotransformed with the CouAA-RS (BBa_K2201204). Maximal absorption of CFP at 430 nm (gray line). Maximal emission of CFP at 475 nm (blue line). Maximal emission of YFP at 525 nm (yellow line).

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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]