Fig 1.
Chromosome vector (ChroV) system in E. coli JM109(DE3) cells.
The scheme of the ChroV-JM109(DE3) system containing the T7 promoter and a silent chloramphenicol resistance gene (cat) that can be activated by homologous recombination with target DNA. GFPuv gene containing H1 and H2 of an RBS plus ATG sequence was inserted into the Fʹ plasmid in E. coli, and the GFPuv gene was expressed and the chloramphenicol resistance gene was activated.
Fig 2.
Expression of GFPuv using the ChroV system.
A. The E. coli cells expressing GFPuv in the Fʹ plasmid (ChroV-GFPuv) and GFPuv in the pET plasmid (pRMT-GFPuv) was observed by colony fluorescent image analysis. B. Growth curves of ChroV and pRMT cells in LB media containing 0.5 mM IPTG and 25 μg/mL kanamycin. C. ChroV and pRMT cells were inoculated in M9 media containing 0.5 mM IPTG. The cell growths were monitored in 96 well plates for 24 h at 37°C with shaking at 200 rpm using the Infinite® 200 PRO microplate reader (Tecan Group Ltd, Switzerland) following the manufacturer’s instructions. Error bars indicate a deviation in the triplicate experiments.
Fig 3.
SDS page analysis of GFPuv expression in ChroV-GFPuv (A) and pRMT-GFPuv (B). 1: cells of OD600 = 0.1; 2: 12 h induction with 0.5 mM IPTG at OD600 = 0.1; 3: cells of OD600 = 0.5; 4: 12 h induction with 0.5 mM IPTG at OD600 = 0.5; 5: cells of OD600 = 1; 6: 12 h induction with 0.5 mM IPTG at OD600 = 1. The cell lysates were loaded for equal protein concentrations as 5 μg in each lane.
Fig 4.
Cloning of the β-carotene synthetic gene cluster in the ChroV system.
Shown are E. coli colonies expressing the β-carotene synthetic genes, which produced yellow-pigmented colonies in the pRMT system [19] and ChroV system (in this study). The ChroV system showed more homogeneous sizes, shapes, and colors for the colonies.
Fig 5.
Stable maintenance of GFPuv-expression levels in conditions without antibiotics.
A. The expression of GFPuv was analyzed by fluorescent intensity at 510 nm after excitation at 385 nm. B. SDS-PAGE analysis of pRMT-GFPuv and ChroV-GFPuv. N: Negative control. 1: E. coli culture time for 4 h; 2: culture time for 33 h; 3: culture time for 67 h; 4: culture time for 100 h; 5: culture time for 134 h; 6: culture time for 168 h. GFPuv indicated by arrow.