Part:BBa_K2556011

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 3356
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 5635
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE 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.