Difference between revisions of "Part:BBa K3081055"
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Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri BBa_K3081007). We found an big decrease in average nucleo-cytoplasmic ratio when treated with CRISPRri(BBa_K3081007)targeted to OriC. In order to extend the CRISPRri(BBa_K3081007) system to other boxes which are shown to have over-inhibition on cell growth and small dynamic range, we improve the system to weaken its effect by adding a degradation signal peptide ssrA to dCas9. This largely accelerates the degradation rate of dCas9 and thus weaken its effect. Again, the CRISPRi system provides solid evidence for retention of dCas9 binding ability and degradation-promoting effect of ssrA. As a matter of fact, CRISPRi system with sgRNA targeted to mRFP coding region shows a more gentle decrease in fluorescence when dCas9 is fused with ssrA tag, while non-binding dCas9 with or without ssrA has no affect on mRFP expression. We tested the effect of the improved CRISPRri-ssrA system with target site to boxes which are shown to have excessive inhibition on cell growth, and found that the effect becomes much milder, which allows for a wider adjusting range. | Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri BBa_K3081007). We found an big decrease in average nucleo-cytoplasmic ratio when treated with CRISPRri(BBa_K3081007)targeted to OriC. In order to extend the CRISPRri(BBa_K3081007) system to other boxes which are shown to have over-inhibition on cell growth and small dynamic range, we improve the system to weaken its effect by adding a degradation signal peptide ssrA to dCas9. This largely accelerates the degradation rate of dCas9 and thus weaken its effect. Again, the CRISPRi system provides solid evidence for retention of dCas9 binding ability and degradation-promoting effect of ssrA. As a matter of fact, CRISPRi system with sgRNA targeted to mRFP coding region shows a more gentle decrease in fluorescence when dCas9 is fused with ssrA tag, while non-binding dCas9 with or without ssrA has no affect on mRFP expression. We tested the effect of the improved CRISPRri-ssrA system with target site to boxes which are shown to have excessive inhibition on cell growth, and found that the effect becomes much milder, which allows for a wider adjusting range. | ||
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| + | https://2019.igem.org/wiki/images/b/b6/T--Peking--R1%2BssrA.gif | ||
| + | R1+ ssrA | ||
| + | https://2019.igem.org/wiki/images/4/43/T--Peking--R1%2Bgif.gif | ||
| + | R1+ | ||
Revision as of 09:07, 21 October 2019
dCas9-ssrA: a fast degradation tag "ssrA" linked to the C-terminal of dCas9
Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri BBa_K3081007). We found an big decrease in average nucleo-cytoplasmic ratio when treated with CRISPRri(BBa_K3081007)targeted to OriC. In order to extend the CRISPRri(BBa_K3081007) system to other boxes which are shown to have over-inhibition on cell growth and small dynamic range, we improve the system to weaken its effect by adding a degradation signal peptide ssrA to dCas9. This largely accelerates the degradation rate of dCas9 and thus weaken its effect. Again, the CRISPRi system provides solid evidence for retention of dCas9 binding ability and degradation-promoting effect of ssrA. As a matter of fact, CRISPRi system with sgRNA targeted to mRFP coding region shows a more gentle decrease in fluorescence when dCas9 is fused with ssrA tag, while non-binding dCas9 with or without ssrA has no affect on mRFP expression. We tested the effect of the improved CRISPRri-ssrA system with target site to boxes which are shown to have excessive inhibition on cell growth, and found that the effect becomes much milder, which allows for a wider adjusting range.
R1+ ssrA
R1+
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1099
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3378
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]