Figure 3

Markerless deletion in C. glutamicum ATCC14067. (a) ΔArgR-cassette construction. The ΔArgR-cassette contains ArgR-L (argR homology left and the 34 bp lox71 sequence), Cre-Kan cassette and ArgR-R (34 bp lox66 sequence and argR right homology). (b) ΔArgR-cassette recombineering with RecET. The primer pairs of ΔargR-JD-S/A were used for the verification of colony PCR. WT is C. glutamicum ATCC14067. (c) Cre excision by the addition of 1 mM theophylline: at least four recombinants were used to evaluate the excision efficiency. (d) Identification markerless deletion of the argR gene. MargR represents the strain in which the argR gene is replaced by the 34 bp sequence of lox72. (e) Identification markerless deletion of the crtB gene in MargR. MargR-crtB represents the strain in which the argR and crtB genes are replaced by the sequence of 34 bp lox72. PR is the ΔargR-JD-S/A primer pair. PC is the ΔcrtB-JD-S/A primer pair. All primers are listed in Supplementary Table S3. Km^r, kanamycin resistance. Cm^r, chloramphenicol resistance.