Fig. 1

Verification of an engineered sequence in the chromosome. a The scheme depicts the changes at the recombineering site to create the motAB gene knockout strain (ΔmotAB) using a chloramphenicol resistance gene (CmR). The positions of the flanking primers for the motAB region (PmotABfw and PmotABrv) are marked, and the corresponding product lengths from PCR are indicated at the bottom. b PCR results of the colonies obtained through recombineering. In lane M, 10 μL of DNA ladder was loaded. In lanes 1–16, 10 μL of PCR products from selected, individual ΔmotAB colonies were loaded (ΔmotAB1 to ΔmotAB16). The PCR products of the control AB1157 strain were added in wells C1 and C2. PCR products of the intended sizes are visible for all 16 selected colonies (~1.1 kbp marked with red arrow) as well as the AB1157 colonies (~1.9 kbp). c A representative DNA sequencing result of the ΔmotAB10 strain at the sites of integration and the corresponding alignment with the expected template DNA sequence are shown. d The important steps of making the Southern blot probes are illustrated. A 650 bp PCR product is amplified from the template plasmid pKD3 and is then labelled with alkaline phosphatase to probe the CmR region (expected size: 6 kbp). e The ethidium bromide stained gel containing the DNA ladder (lane M), the restriction-digested AB1157 genome (lane 1), restriction-digested genomes of two ΔmotAB colonies (lane 2: ΔmotAB10 and lane 3: ΔmotAB14 which were verified by PCR and DNA sequencing). The Southern blot results show that the AB1157 sample in lane 1 has no insert, as expected; lane 2 with ΔmotAB10 has one band (6 kbp) at the right fragment size showing that the integration was successful at the predicted site; lane 3 with ΔmotAB14 has two bands (6 kbp and 2 kbp). f The growth of ΔmotAB10 and ΔmotAB14 strains in 96 well-plate reader containing LB medium with various concentrations of chloramphenicol (17 μg/mL to 68 μg/mL). The results show that ΔmotAB14 strain containing the extraneous insertion grew at a higher concentration of chloramphenicol (51 μg/mL) than the normal concentration (34 μg/mL), while the ΔmotAB10 did not grow at 51 μg/mL of chloramphenicol