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. 2019 Feb 12;9:1839. doi: 10.1038/s41598-018-37949-x

Figure 2.

Figure 2

Heterologous expression of Cas9 in L. biflexa cells and nuclease activity validation in E. coli. (A) Map of the plasmids obtained in this work, depicting important features and unique restriction sites. (B) Imunoblots of E. coli and L. biflexa containing wild type pMaOri (1), pMaOriCas9 (2) or pMaOri.p32Cas9 (3) with anti-Cas9 and anti-DnaK antibodies. Data of E. coli and L. biflexa come from different blottings. (C) Transformation efficiency of E. coli cells containing plasmids pMaOriCas9, pMaOri.p32Cas9 or no plasmid with plasmids pGKLep4 (wild type) or pGKLep4 containing J23119 promoter directing the transcription of sgRNA targeting the E. coli βgal gene (pGKLep4sgRNAbgalEcoli) or an unrelated sequence (leptospiral β-galactosidase, pGKLep4sgRNAbgal), as control. After transformation, cells were seeded onto LB plates supplemented with spectinomycin and kanamycin. As a control, wild type cells transformed with pGKLep4 plasmids were seed in LB plus kanamycin plates. Cell counting are presented as average plus standard deviation of 3 independent experiments.