Difference between revisions of "Team:Bielefeld-CeBiTec/Results/toolbox/photolysis"

Line 28: Line 28:
 
</div>
 
</div>
  
−
 
+
<div class="contentbox">
 +
<div class="bevel tr"></div>
 +
<div class="content">
 +
<h2> Cloning of this NPA-RS in pSB1C3 and pSB3T5</h2>
 +
<article>
 +
We then used the protein sequence of the clone with the highest fidelity for 2-NPA and translated it into a gene sequence which was then codon optimized for E.coli. We designed it with matching overhangs of 35bp to a linearized ONBY-Part (K1416000) in pSB1C3 to get the sequence in a matching expression cassette. The psb1c3 backbone is a high copy plasmid and for an adequate usage of the aaRS it is needed on a low copy plasmid. We so used BioBrick assembly to get the insert of the new 2-NPA-Part (K2201200) in the low copy plasmid of pSB1K3 (Figure 2) for further use. The Insert of K2201200 in the low copy plasmid is available on request at the CeBiTec.
 +
</article>
 +
<div class="figure small">
 +
<img class="figure image" src="https://static.igem.org/mediawiki/2017/6/6c/T--Bielefeld-CeBiTec--YKE_NPA-RS_in_psb1c3_psb3k5.png">
 +
<p class="figure subtitle"><b>Figure 2: Two Plasmids we created for our toolkit for the iGEM community. Left: 2-NPA-RS in the pSB1C3 high copy plasmid (K2201200). Right: 2-NPA-RS in the pSB3T5 low copy plasmid (available on request) at the CeBiTec.</b><p>
 +
</div>
 +
</div>
 +
<div class="bevel bl"></div>
 +
</div>
  
  

Revision as of 15:11, 4 October 2017

Photolysis

Design of 2-NPA-RS

At first, we designed a gene synthesis at IDT based on the selection experiment from Peters et al. (2009) to get an aminoacyl-tRNA-synthetase to incorpate 2-nitrophenylalanine. An alignment of the protein sequences of the native M. jannaschii TyrRS which was the basis of the selection experiment and the used 2-Nitrophenylalanine-synthetase is shown in Figure 1. They differ in ten amino acids.

Figure 1: Alignment of the protein sequences of the M. jannaschii tyrosyl synthetase and the 2-Nitrophenylalanine synthetase designed by Peters et al.

Cloning of this NPA-RS in pSB1C3 and pSB3T5

We then used the protein sequence of the clone with the highest fidelity for 2-NPA and translated it into a gene sequence which was then codon optimized for E.coli. We designed it with matching overhangs of 35bp to a linearized ONBY-Part (K1416000) in pSB1C3 to get the sequence in a matching expression cassette. The psb1c3 backbone is a high copy plasmid and for an adequate usage of the aaRS it is needed on a low copy plasmid. We so used BioBrick assembly to get the insert of the new 2-NPA-Part (K2201200) in the low copy plasmid of pSB1K3 (Figure 2) for further use. The Insert of K2201200 in the low copy plasmid is available on request at the CeBiTec.

Figure 2: Two Plasmids we created for our toolkit for the iGEM community. Left: 2-NPA-RS in the pSB1C3 high copy plasmid (K2201200). Right: 2-NPA-RS in the pSB3T5 low copy plasmid (available on request) at the CeBiTec.