Difference between revisions of "Team:Bielefeld-CeBiTec/Improve"

Line 12: Line 12:
 
</div>
 
</div>
 
</div>
 
</div>
 +
 +
<div class="contentbox">
 +
<div class="bevel tr"></div>
 +
<div class="content">
 +
<article>
 +
<b>Note:</b> For all absorption and emission measurements mentioned further on, we used the Tecan Reader infinite M200. All measurements were made by one of the team members.
 +
</article>
 +
</div>
 +
<div class="bevel bl"></div>
 +
</div>
 +
 +
<div class="contentbox">
 +
<div class="bevel tr"></div>
 +
<div class="content">
 +
<h2>Introduction</h2>
 +
<article>
 +
Besides the integration of unnatural bases into the genetic code of <i>E.coli</i> and the usage of non-canonical amino acids (ncAA) for advanced protein design, the creation and selection of new aminoacyl-tRNA synthetases (aaRS) is a fundamental part of our project. To determine the most effective and specific aaRS for the incorporation of the desired ncAAs, an evaluation system of the synthetases seemed meaningful to us. We so got aware of the synthetase test system “pFRY” (a Reporter Plasmid for Measuring ncAA Incorporation, BBa_K1416004) of the iGEM-Team Austin, Texas, 2014 which was also used by the team Aachen 2016 (BBa_K2020040).
 +
The pFRY plasmid consists of an 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.
 +
We liked this idea very much but 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.
 +
</article>
 +
</div>
 +
<div class="bevel bl"></div>
 +
</div>
 +
 +
<div class="contentbox">
 +
<div class="bevel tr"></div>
 +
<div class="content">
 +
<h2>Short summary</h2>
 +
<article>
 +
We designed the CFP-YFP test system (BBa_K2201343) just like the RFP-GFP system and only changed the coding sequences of the both fluorescent proteins.
 +
First we compared the CFP and the RFP domain of the test system by transforming them into BL21(DE3) solely and performing some absorption and emission measurements. We detected that the absorption and the emission maxima of the RFP are equal to the information of Texas 2014 but they are only 20 nm apart, which lead to the first measurement problems. Furthermore we detected a second absorption maximum at 505 nm, not mentioned by Texas 2014. It was more suitable for exciting RFP but on the other hand very close to the absorption maximum of GFP (485 nm) which leads to a heavy excitement and emission of RFP when we wanted to excite only the GFP. The absorption and emission maxima of CFP are ~ 45 nm apart and so very sharp to differentiate. Also we could detect that the emission signal of the CFP is approximately five times higher than of RFP, if both of them are excited at their absorption maxima under comparable conditions. We could replicate there results <i>in vitro</i> and <i>in vivo</i>.
 +
We proofed that our FRET system if functional, for there were high YFP-signals of the fusion protein detected, if excited at 475 nm, specific for YFP excitation, and when excited at 430 nm, specific for CFP excitation. This means that the energy of the CFP emission is transferred to the YFP and thus the FRET system is verified. Also there was a still a clear CFP signal present at 475 nm which means that only the whole fusion protein of CFP and YFP forms the FRET system, and that solely CFP, where no ncAA was incorporated in the linker, still is present and detectable alongside with the FRET system.
 +
We so developed ne new ranking system for the quality of synthetases, slightly variated form the system of Texas 2014, matching for our improved FRET system.
 +
</article>
 +
</div>
 +
<div class="bevel bl"></div>
 +
</div>
 +
 +
  
 
</div>
 
</div>

Revision as of 14:22, 26 October 2017

Part Improvement
Note: For all absorption and emission measurements mentioned further on, we used the Tecan Reader infinite M200. All measurements were made by one of the team members.

Introduction

Besides the integration of unnatural bases into the genetic code of E.coli and the usage of non-canonical amino acids (ncAA) for advanced protein design, the creation and selection of new aminoacyl-tRNA synthetases (aaRS) is a fundamental part of our project. To determine the most effective and specific aaRS for the incorporation of the desired ncAAs, an evaluation system of the synthetases seemed meaningful to us. We so got aware of the synthetase test system “pFRY” (a Reporter Plasmid for Measuring ncAA Incorporation, BBa_K1416004) of the iGEM-Team Austin, Texas, 2014 which was also used by the team Aachen 2016 (BBa_K2020040). The pFRY plasmid consists of an 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. We liked this idea very much but 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.

Short summary

We designed the CFP-YFP test system (BBa_K2201343) just like the RFP-GFP system and only changed the coding sequences of the both fluorescent proteins. First we compared the CFP and the RFP domain of the test system by transforming them into BL21(DE3) solely and performing some absorption and emission measurements. We detected that the absorption and the emission maxima of the RFP are equal to the information of Texas 2014 but they are only 20 nm apart, which lead to the first measurement problems. Furthermore we detected a second absorption maximum at 505 nm, not mentioned by Texas 2014. It was more suitable for exciting RFP but on the other hand very close to the absorption maximum of GFP (485 nm) which leads to a heavy excitement and emission of RFP when we wanted to excite only the GFP. The absorption and emission maxima of CFP are ~ 45 nm apart and so very sharp to differentiate. Also we could detect that the emission signal of the CFP is approximately five times higher than of RFP, if both of them are excited at their absorption maxima under comparable conditions. We could replicate there results in vitro and in vivo. We proofed that our FRET system if functional, for there were high YFP-signals of the fusion protein detected, if excited at 475 nm, specific for YFP excitation, and when excited at 430 nm, specific for CFP excitation. This means that the energy of the CFP emission is transferred to the YFP and thus the FRET system is verified. Also there was a still a clear CFP signal present at 475 nm which means that only the whole fusion protein of CFP and YFP forms the FRET system, and that solely CFP, where no ncAA was incorporated in the linker, still is present and detectable alongside with the FRET system. We so developed ne new ranking system for the quality of synthetases, slightly variated form the system of Texas 2014, matching for our improved FRET system.