Difference between revisions of "Part:BBa K3081060"
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A double-input CRISPRi system with tunable dCas9 and sgRNA targeting low-affinity DnaA box | A double-input CRISPRi system with tunable dCas9 and sgRNA targeting low-affinity DnaA box | ||
Unlike traditional CRISPRi system, our new system is more controllable with multiplex inputs. The expression of dCas9 is driven by an arabinose-inducible promoter with low leakage and great switch property. The expression of sgRNA is driven by a novel inducible T7 system, in which IPTG triggers T7 polymerase expression and the sgRNA expression increases with the T7 polymerase expression level. The sgRNA targets a DNA sequence in E. coli OriC called M box, which has low affinity to DnaA, a key factor in E. coli genome replication initiation. Cell growth rate, morphology and nuclear-cytoplasmic was measured, which proved that DNA replication can controlled and the control is of large dynamic range, We co-transformed this part with GFP, indigo and polyhydroxyalkanoates (PHA) production plasmids and found it can enhance cell function. It has great application prospect in not only industrial production but also bacteria therapies. Engineered bacteria can be used in a highly controllable manner. Risks of systemic infection caused by bacteria overgrowth can be reduced. | Unlike traditional CRISPRi system, our new system is more controllable with multiplex inputs. The expression of dCas9 is driven by an arabinose-inducible promoter with low leakage and great switch property. The expression of sgRNA is driven by a novel inducible T7 system, in which IPTG triggers T7 polymerase expression and the sgRNA expression increases with the T7 polymerase expression level. The sgRNA targets a DNA sequence in E. coli OriC called M box, which has low affinity to DnaA, a key factor in E. coli genome replication initiation. Cell growth rate, morphology and nuclear-cytoplasmic was measured, which proved that DNA replication can controlled and the control is of large dynamic range, We co-transformed this part with GFP, indigo and polyhydroxyalkanoates (PHA) production plasmids and found it can enhance cell function. It has great application prospect in not only industrial production but also bacteria therapies. Engineered bacteria can be used in a highly controllable manner. Risks of systemic infection caused by bacteria overgrowth can be reduced. | ||
Latest revision as of 09:47, 21 October 2019
pBAD-dCas9-T7-M+
A double-input CRISPRi system with tunable dCas9 and sgRNA targeting low-affinity DnaA box Unlike traditional CRISPRi system, our new system is more controllable with multiplex inputs. The expression of dCas9 is driven by an arabinose-inducible promoter with low leakage and great switch property. The expression of sgRNA is driven by a novel inducible T7 system, in which IPTG triggers T7 polymerase expression and the sgRNA expression increases with the T7 polymerase expression level. The sgRNA targets a DNA sequence in E. coli OriC called M box, which has low affinity to DnaA, a key factor in E. coli genome replication initiation. Cell growth rate, morphology and nuclear-cytoplasmic was measured, which proved that DNA replication can controlled and the control is of large dynamic range, We co-transformed this part with GFP, indigo and polyhydroxyalkanoates (PHA) production plasmids and found it can enhance cell function. It has great application prospect in not only industrial production but also bacteria therapies. Engineered bacteria can be used in a highly controllable manner. Risks of systemic infection caused by bacteria overgrowth can be reduced.
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 6316 - 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 AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Illegal SapI.rc site found at 5369