Difference between revisions of "Part:BBa K786031:Experience"

 
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===Applications of BBa_K786031===
 
===Applications of BBa_K786031===
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'''Expected Result'''
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Firstly, we need to test if the proposed system present in the E. coli. We Used primers to amplify the Cas3 and cascade proteins from E. coli K12 genome DNA. Expected band size of cas3 2.7kbp and cascade 4.4kbp are shown in the gel photo, and sequencing has be done, which proves the existence of these proteins in its genome, thus we can utilize this CRISPR/Cas3 system as a tool for our new safety approach. In the future we can make them into biobrick format and apply into microorganism other than E. coli.
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[[Image:https://static.igem.org/mediawiki/2012/d/d5/Iii.png]]
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To evaluate whether the RSR system transformed into E. coli can degrade genomic DNA and eventually lead to cell death, we measured OD600 of cell cultures every 0.5 h and plotted the growth curves of transformed E. coli (indicated as solid lines) with or without 0.4 mM IPTG activation. Paired t-test showed that there is no significant difference in OD600 throughput the whole experiment with or without RSR activation by IPTG.
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[[Image:https://static.igem.org/mediawiki/2012/3/3c/Safeapp.png]]
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The result agrees with the percentage of survival (indicated as dashed lines) derived from live/dead fluorescent staining assay (Invitrogen) measuring bacterial apoptotic-like rate, which showed no difference in survival percentage with or without RSR activation. It was expected that the survival percentage drops significantly after IPTG induction. Further optimization of design of the RSR region and experimental conditions is required for more specific targeting.
  
 
===User Reviews===
 
===User Reviews===

Revision as of 07:26, 28 September 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K786031

Expected Result 


Firstly, we need to test if the proposed system present in the E. coli. We Used primers to amplify the Cas3 and cascade proteins from E. coli K12 genome DNA. Expected band size of cas3 2.7kbp and cascade 4.4kbp are shown in the gel photo, and sequencing has be done, which proves the existence of these proteins in its genome, thus we can utilize this CRISPR/Cas3 system as a tool for our new safety approach. In the future we can make them into biobrick format and apply into microorganism other than E. coli.

File:Https://static.igem.org/mediawiki/2012/d/d5/Iii.png 


To evaluate whether the RSR system transformed into E. coli can degrade genomic DNA and eventually lead to cell death, we measured OD600 of cell cultures every 0.5 h and plotted the growth curves of transformed E. coli (indicated as solid lines) with or without 0.4 mM IPTG activation. Paired t-test showed that there is no significant difference in OD600 throughput the whole experiment with or without RSR activation by IPTG.

File:Https://static.igem.org/mediawiki/2012/3/3c/Safeapp.png 


The result agrees with the percentage of survival (indicated as dashed lines) derived from live/dead fluorescent staining assay (Invitrogen) measuring bacterial apoptotic-like rate, which showed no difference in survival percentage with or without RSR activation. It was expected that the survival percentage drops significantly after IPTG induction. Further optimization of design of the RSR region and experimental conditions is required for more specific targeting.

User Reviews

UNIQ5cd40f3852f948d4-partinfo-00000000-QINU UNIQ5cd40f3852f948d4-partinfo-00000001-QINU