Part:BBa_K2556000

AraC-Pbad-gfp-Cas9

Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes. Apart from its original function in bacterial immunity, the Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double strand breaks in DNA. In this part, Cas9's expression can be induced by Ara. Upstream of cas is gfp, which can be used to indicate whether gene is normally transcribed.

Usage and Biology

The arabinose-induced promoter offers the possibility to regulate the expression of Cas9 protein in Escherichia coli by adding or not adding a certain amount of arabinose into the culture, therefore control the CRISPR/Cas system.

Characterize

We built this part on pGLO and named it pGLO-Cas9. To verify whether the expressed cas9 protein has the function of cutting the target gene, we used E. coli MG1655 as a chassis. In addition, we will use the pTF plasmid to transcribe the sgRNA that targets the panD gene. Since the original pTF is not compatible with pGLO, we have built a pTF-p15A that is compatible with pGLO. Finally, we characterized the function of the Cas9 gene based on the growth curve of the bacteria.
When the function of Cas9 was verified, we replaced the sgRNA targeting panD with the sgRNA targeting the chloramphenicol gene. The plasmid we constructed was named pTF-p15A-cm. And we used E. coli MG1655 containing the chloramphenicol gene on genome for validation experiments.
*panD gene is a gene on the genome of E. coli, in the previous research of our lab, we obtained a panD-deficient strain of E. coli.

Experimental Results

Fig.1 Growth curve, add 10mM ara after 3 hours of culture

Fig.2 Growth curve, add ara after 5 hours of culture

In our experiment we required transformation data and two growth curve in the end. Fig.1 shows that after adding arabinose, E. coli MG1655 with PBAD-Cas9 plasmid stopped growing. We use OD600 to characterize the cell amount proving that the expression of Cas9 protein can be regulated by 10mM arabinose. Fig.2 shows how different concentration of arabinose affects the Cas9 expression.
In the chloramphenicol gene cleavage experiment, we used a plate containing Ara for transformation experiments, and the cleavage of the chloramphenicol gene can be characterized by transformation efficiency. The results are shown in Table 1:

Table1. Number of transformants

Our results show that inducing the expression of cas9 by ara has no effect on the transformation efficiency of wild type, and the transformation efficiency of E.coli cmR is greatly reduced. Therefore, we can conclude that the transformation efficiency is reduced because Cas9 cleaves the chloramphenicol gene on the genome under the guidance of sgRNA-cm. Moreover, when we did not add Ara, the transformation efficiency of E.coli cmR was also lower than that of E.coli wild type. This suggests that Cas9 exhibits high gene cleavage efficiency in bacteria because PBAD is a well-regulated promoter.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1252
  Illegal NheI site found at 3092
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 1771
  Illegal BamHI site found at 5371
  Illegal XhoI site found at 1672
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

References

[1]Jiang W, Bikard D, Cox D, et al. CRISPR-assisted editing of bacterial genomes[J]. Nature Biotechnology, 2013, 31(3):233-239.
[2]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.
[3]Didovyk A, Borek B, Hasty J, et al. Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9[J]. Acs Synthetic Biology, 2016, 5(1):81-88.
[4]Doench J G, Fusi N, Sullender M, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9[J]. Nature Biotechnology, 2016, 34(2):184-191.