Fig. 2.

Point mutation in the promoter resulted in enhanced expression of OmpF. a Sequences of the OmpF promoter − 80 region. The black box indicates the mutation site that differs between the normal ER2566 strain and the OmpF-constitutive-expression strain ER0808. b Point mutation strategy utilizing CRISPR/Cas9-coupled lambda Red recombineering. The sequence corresponding to the ompF protospacer is shown in blue, and the sequence corresponding to the − 77 locus is shown in red. “Rep” corresponds to direct repeat sequences necessary for crRNA processing that flank the targeting spacer. c Representative sequencing results of the OmpF promoter at the − 80 region from edited ER2566 colonies. Of 24 independent colonies randomly picked for editing confirmation, 6 colonies (5#, 14#, 17#, 18#, 22# and 24#) harbored the expected silent Ala130 mutation (corresponding to GCA to GCT) and the 1-bp deletion of a thymidine at the − 77 locus. One colony (6#) suffered a wrong mutation of Ala130 to Val130 (GCA to GTT), and the remaining 17 colonies had no 1-bp deletion of a thymidine. Sequences for 4#, 8# and 9# are shown