Difference between revisions of "Team:BostonU/App 2/Results"

Line 20: Line 20:
 
<p>Unfortunately, the system we designed had some errors, and we were unable to gather any useful data on our split SaCas9 experiment. We created 16 split sites within SaCas9 and cloned most of these constructs into the FKBP/FRB dimerizable domains. We tested the full SaCas9 on the traffic light reporter, and there was no fluorescence at all, so we concluded that no double stranded break had been made. We realize that there may have been an issue with our choice of target sequence within the traffic light reporter. For future analysis, we can test the SpCas9 on the traffic light reporter to see how feasible the system is before moving on to SaCas9 and split SaCas9 halves.</p>
 
<p>Unfortunately, the system we designed had some errors, and we were unable to gather any useful data on our split SaCas9 experiment. We created 16 split sites within SaCas9 and cloned most of these constructs into the FKBP/FRB dimerizable domains. We tested the full SaCas9 on the traffic light reporter, and there was no fluorescence at all, so we concluded that no double stranded break had been made. We realize that there may have been an issue with our choice of target sequence within the traffic light reporter. For future analysis, we can test the SpCas9 on the traffic light reporter to see how feasible the system is before moving on to SaCas9 and split SaCas9 halves.</p>
  
−
<center><img style="height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/e/e7/Sacas9_gfp.png/800px-Sacas9_gfp.png" />
+
<center><img style="height:40%; width:40%;" src="https://static.igem.org/mediawiki/2015/thumb/e/e7/Sacas9_gfp.png/800px-Sacas9_gfp.png" />
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
−
<img style= "height:25%; width:25%;" src="https://static.igem.org/mediawiki/2015/thumb/e/e4/Sacas9_mcherry.png/800px-Sacas9_mcherry.png" />
+
<img style= "height:40%; width:40%;" src="https://static.igem.org/mediawiki/2015/thumb/e/e4/Sacas9_mcherry.png/800px-Sacas9_mcherry.png" />
 
<center>
 
<center>
  

Revision as of 21:37, 18 September 2015

Motivation Design Results

Results

Unfortunately, the system we designed had some errors, and we were unable to gather any useful data on our split SaCas9 experiment. We created 16 split sites within SaCas9 and cloned most of these constructs into the FKBP/FRB dimerizable domains. We tested the full SaCas9 on the traffic light reporter, and there was no fluorescence at all, so we concluded that no double stranded break had been made. We realize that there may have been an issue with our choice of target sequence within the traffic light reporter. For future analysis, we can test the SpCas9 on the traffic light reporter to see how feasible the system is before moving on to SaCas9 and split SaCas9 halves.

               

Towards the end of our project, the Feng Zhang group at MIT published the crystal structure of saCas9 in Cell1. The paper demonstrated the development of a conditionally dimerizable saCas9 system using the FKBP-FRB domain pairs and PYL-ABI domain pairs, which is what we wanted to accomplish.

One facet worth noting is that the Zhang group used the 3D structure of the saCas9 protein to target flexible linker regions that would serve as potential split sites. Out of the three split sites that they chose, the one that worked best was almost identical (one amino acid away) to one of the split sites that we identified using our model. Furthermore, the other two split sites that they chose which were not as successful happened to be located in regions with secondary structural elements.

This important result gave us some experimental validation that our model could offer scientists insights into choosing promising viable split sites for proteins, without only relying on 3D structures of proteins.

Citations

  1. Nureki, Osamu et al., “Crystal structure of staphylococcus aureus Cas9”, Cell, 2015.