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BBa_K2556000

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 engineered 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.

Experiment design

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

In our experiment we required transformation data and two growth curves in the end. Fig.1 shows that after adding arabinose, E. coli MG1655 with P_BAD-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:

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 cm^R 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 cm^R was also lower than that of E.coli wild type. This suggests that Cas9 exhibits high gene cleavage efficiency in bacteria because P_BAD is a well-regulated promoter.

BBa_K2556011

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 P_fixk2 promoter. In blue light(480 nm) YF1 don’t phosphorylate FixJ response regulator, so P_fixk2 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).Firstly, 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.

BBa_K2556333

Under dark conditions, the phosphate group is transferred from Yf1 protein to FixJ protein, the phosphorylated FixJ protein activates the P_fixK2 promoter and then activates the downstream gene expression. When induced by light, the phosphorylation of Fixj protein will be blocked, and the expression of genes regulated by P_fixK2 promoters will be inhibited.

Characterize

To further verify the effectiveness of the system, the plasmids with the system were transferred into different hosts and cultured for more than 24 hours under blue light and dark light respectively. The fluorescence value of eGFP in the culture medium was measured.

Experimental Results

Detection of expression efficiency of primary light control system in different hosts

The dusk-eGFP-pUC57 plasmid was transferred into the host of DH5α,BL21,4MG1655 and BW25113, cultured under dark or blue light for a period of time and the fluorescence value of eGFP was detected. After 24 hours, the fluorescence value of the experimental group in the dark culture showed an obvious increasing trend. There was no significant increase in fluorescence value in both the experimental group under blue light and the negative control group.