Difference between revisions of "Part:BBa K3081041"
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+ | __NOTOC__ | ||
+ | <partinfo>BBa_K3081041 short</partinfo> | ||
+ | This composite part uses a CRISPR-based DNA replication interference system for the copy number control of the p15A plasmid. The dCas9 is expressed in an inducible manner with a ssrA degradation tag fused to its C-terminal to minimize the basal expression. The sgRNA has a 20-bp complementary sequence with the top strand (complementary to the strand that replication activator (RNA II) binds to) of initiation site (+1) on p15A origin. It can efficiently downregulate the plasmid copy number but has some unexpected leackage. | ||
+ | <center> | ||
+ | https://2019.igem.org/wiki/images/c/ca/T--Peking--p15A_mechanism.png | ||
+ | </center> | ||
+ | <center> | ||
+ | <b>Design of the p15A plasmid copy number control system</b> | ||
+ | </center> | ||
+ | <center> | ||
+ | https://2019.igem.org/wiki/images/f/fa/T--Peking--p15A_result.png | ||
+ | </center> | ||
+ | <center> | ||
+ | <b>The effect of 4 target sites on p15A origin</b> | ||
+ | </center> | ||
+ | |||
+ | |||
+ | Reference: | ||
+ | |||
+ | [1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735. | ||
+ | |||
+ | |||
+ | |||
+ | <!-- Add more about the biology of this part here | ||
+ | ===Usage and Biology=== | ||
+ | |||
+ | <!-- --> | ||
+ | <span class='h3bb'>Sequence and Features</span> | ||
+ | <partinfo>BBa_K3081041 SequenceAndFeatures</partinfo> | ||
+ | |||
+ | |||
+ | <!-- Uncomment this to enable Functional Parameter display | ||
+ | ===Functional Parameters=== | ||
+ | <partinfo>BBa_K3081041 parameters</partinfo> | ||
+ | <!-- --> |
Latest revision as of 19:19, 21 October 2019
CRISPR-based replication interference system for p15A plasmid copy number control, ini(-) site
This composite part uses a CRISPR-based DNA replication interference system for the copy number control of the p15A plasmid. The dCas9 is expressed in an inducible manner with a ssrA degradation tag fused to its C-terminal to minimize the basal expression. The sgRNA has a 20-bp complementary sequence with the top strand (complementary to the strand that replication activator (RNA II) binds to) of initiation site (+1) on p15A origin. It can efficiently downregulate the plasmid copy number but has some unexpected leackage.
Design of the p15A plasmid copy number control system
The effect of 4 target sites on p15A origin
Reference:
[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
Illegal NheI site found at 2321
Illegal NheI site found at 5495
Illegal NheI site found at 5518 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
Illegal BamHI site found at 4600 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 5522
Illegal AgeI site found at 979 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961