Difference between revisions of "Part:BBa K2556011"
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YF1 is a fusion protein of a LOV protein domain and histidine kinase. In the dark YF1 phosphorylates FixJ response regulator and phospholylated FixJ response regulator activates Pfixk2 promoter. In blue light(480 nm) YF1 don’t phosphorylate FixJ response regulator, so Pfixk2 promoter isn’t activated. | YF1 is a fusion protein of a LOV protein domain and histidine kinase. In the dark YF1 phosphorylates FixJ response regulator and phospholylated FixJ response regulator activates Pfixk2 promoter. In blue light(480 nm) YF1 don’t phosphorylate FixJ response regulator, so Pfixk2 promoter isn’t activated. | ||
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===<h1>Characterize</h1>=== | ===<h1>Characterize</h1>=== | ||
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<p>Figure 1. Results of transformation experiments</p> | <p>Figure 1. Results of transformation experiments</p> | ||
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<partinfo>BBa_K2556011 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2556011 SequenceAndFeatures</partinfo> | ||
===<h1>References</h1>=== | ===<h1>References</h1>=== | ||
| − | 1 | + | [1]Wang G, Lu X, Zhu Y, et al. A light-controlled cell lysis system in bacteria.[J]. Journal of Industrial Microbiology & Biotechnology, 2018:1-4. |
| − | <br>2 | + | <br>[2]Wu H, Wang Y, Wang Y, et al. Quantitatively relating gene expression to light intensity via the serial connection of blue light sensor and CRISPRi[J]. Acs Synthetic Biology, 2014, 3(12):979. |
| − | <br>3 | + | <br>[3]Citorik R J, Mimee M, Lu T K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases[J]. Nature Biotechnology, 2014, 32(11):1141-1145. |
| + | <br>[4]Jiang W, Bikard D, Cox D, et al. CRISPR-assisted editing of bacterial genomes[J]. Nature Biotechnology, 2013, 31(3):233-239. | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K2556011 parameters</partinfo> | <partinfo>BBa_K2556011 parameters</partinfo> | ||
Latest revision as of 19:26, 17 October 2018
Light-involved system control the expression of Cas9
YF1 is a fusion protein of a LOV protein domain and histidine kinase. In the dark YF1 phosphorylates FixJ response regulator and phospholylated FixJ response regulator activates Pfixk2 promoter. In blue light(480 nm) YF1 don’t phosphorylate FixJ response regulator, so Pfixk2 promoter isn’t activated.
Characterize
The strain we used is E.coli Bl21 ΔpanD,which is a panD mutant, the original panD gene was replaced by the chloramphenicol resistance gene in the genome(cm).First, we need to prepare E.coli Bl21 ΔpanD competent cells and transform dusk-Cas9-pUC57(contain BBa_K2556011) plasmid into it.Then, we need to culture the transformants and prepare them into competent cells, and then transform pTargetF-cm plasmid,pSU20 plasmid (as control) and pTargetF-panD plasmid (as control) into it,respectively.Finally, the transformed plates were placed in blue and dark devices overnight.
Experimental Results
Under light and dark conditions, the transformation efficiencies with pTargetF-panD were 1.185 and 1.295 folds of thoese with pTargetF-cm respectively, demonstrating that pTargetF-cm could guide Cas9 to the cm gene on the genome and resulted in the decrease of transformation efficiency. The result also reflect that the blue light provided cannot completely suppress the expression of CRISPR/Cas9. It is also remarkable that the transformation efficiency with pTargetF-cm under the dark condition was lower than that under the light condition, indicating that the CRISPR/Cas9 system showed stronger activity under dark condition, achieving the purpose of cutting a resistance gene with the light-controlled CRISPR/Cas9 system.
Figure 1. Results of transformation experiments
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 3356 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 5635
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 605
Illegal NgoMIV site found at 677
Illegal NgoMIV site found at 767
Illegal NgoMIV site found at 785
Illegal NgoMIV site found at 1297
Illegal NgoMIV site found at 1590
Illegal NgoMIV site found at 1684
Illegal AgeI site found at 319
Illegal AgeI site found at 1465 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1354
Illegal BsaI.rc site found at 218
References
[1]Wang G, Lu X, Zhu Y, et al. A light-controlled cell lysis system in bacteria.[J]. Journal of Industrial Microbiology & Biotechnology, 2018:1-4.
[2]Wu H, Wang Y, Wang Y, et al. Quantitatively relating gene expression to light intensity via the serial connection of blue light sensor and CRISPRi[J]. Acs Synthetic Biology, 2014, 3(12):979.
[3]Citorik R J, Mimee M, Lu T K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases[J]. Nature Biotechnology, 2014, 32(11):1141-1145.
[4]Jiang W, Bikard D, Cox D, et al. CRISPR-assisted editing of bacterial genomes[J]. Nature Biotechnology, 2013, 31(3):233-239.