Fig. 3

Chromosomal integration of sucrose and lactose catabolism pathways into the E. coli chromosome. a PCR confirmation of the integration of the four DNA fragments and cat-yfp cassette (15 kb DNA) into the fliK locus of the E. coli (EcΔlac) chromosome. 1,2,3,4,cat-yfp: DNA fragments 1,2,3,4 and cat-yfp cassette integrated into EcΔlac(I1234); negative control (EcΔlac); JEc1, J12, J23, J34: junctions between the chromosome and DNA fragment 1, DNA fragments 1 and 2, DNA fragments 2 and 3, DNA fragments 3 and 4 in EcΔlac(I1234), respectively. Expected amplicon sizes are shown on the right. b LB agar with chloramphenicol was used to examine chloramphenicol resistance in EcΔlac (left) and EcΔlac(I1234) (right). Only the engineered strain EcΔlac(I1234) grew on chloramphenicol. c EcΔlac (left) and EcΔlac(I1234) (right) grown on LB plates were investigated for the expression of the integrated yellow fluorescent protein (mVenus) using blue light. EcΔlac(I1234) emitted yellow fluorescent light. d MacConkey agar was used to analyze the ability to ferment lactose in EcΔlac (left) and EcΔlac(I1234) (right). The red color of EcΔlac(I1234) is caused by a pH change resulting from lactose fermentation. e M9 medium supplemented with sucrose (20 mg/ml) was used to investigate the ability to utilize sucrose as a carbon source in EcΔlac (left) and EcΔlac(I1234) (right). EcΔlac(I1234) grew on the minimal sucrose medium