Figure 1.
Strategy for in vivo cloning by the recruitment of the translation initiation sequence for antibiotic marker expression.
(A) The receiver plasmid, pRMT, contains a silent chloramphenicol resistance gene (Cmr) that is activated by homologous recombination with the target DNA in E. coli cells. The DNA insert is comprised of 5'-homologous sequences (H1), target gene sequences, the RBS plus ATG for activation of the silent selection marker, and 3'-homologous sequences (H2). F and R indicate primers with specific sequences for the amplification of the target DNA. PT7 and TT7 represent the T7 promoter and T7 terminator, respectively. (B) Shown are the sequences of the homologous region used to prepare the inserts with the target gene and the region for homologous recombination with the silent chloramphenicol resistance gene in receiver plasmid, pRMT and pRMT-sacB.
Figure 2.
Restored antibiotic resistance and GFPuv expression by homologous recombination.
(A) E. coli JM109 cells containing the pRMT plasmid were sensitive to 25 μg/l of chloramphenicol in LB broth because the chloramphenicol resistance gene is silent in the receiver plasmid. (B) GFPuv expression is observed in the positive colonies. (C) The effect of the amount of DNA insert on in vivo cloning. The DNA inserts were transformed into the cells containing the pRMT vector by electroporation. The colony number increases with higher concentrations of the insert used for electroporation. (D) The effect of the homologous sequence length on the pRMT system.
Figure 3.
Size dependency and the cloning of the carotenoid synthetic gene clusters.
(A) Shown are the genetic structures of the various carotenoid synthetic gene clusters used in this study. PT7 in synthetic gene cluster represent the T7 promoter. P1, P2, and P3 refer to additional promoters. (B) Shown are E. coli colonies expressing the various target genes: the 2-kb insert exhibited green fluorescence, while the 4-kb insert produced yellow-pigmented colonies. The 6-kb and 10-kb inserts showed mixed colonies of yellow and red, the colors of β-carotenoid and astaxanthin, respectively. (C) Analysis of the effect of insert size on the cloning efficiency.
Figure 4.
The homogeneity problem and the solution using a counter selection marker, sacB.
(A) Shown are the genetic structure and scheme of the pRMT and pRMT-sacB plasmids when the GFPuv gene was recombined into both plasmids. (B) Shown is the restriction analysis of the target plasmids from selected hits. upper and lower panels represent the electrophoresis results from the pRMT and pRMT-sacB plasmids, respectively, digested with BglII and ScaI. The C- and C+ represent the indicator bands of one without and one with the insert, respectively while the GFPuv gene was used as a target gene.