Figure 1.
(A) A schematic diagram of the proposed CPEC mechanism for cloning an individual gene. The vector and the insert share overlapping regions at the ends. After denaturation and annealing (Step 1), the hybridized insert and vector extend using each other as a template until they complete a full circle and reach their own 5′-ends (Step 2). The final completely assembled plasmid has two nicks, one on each strand, at the positions marked by an arrow head. They can be used for transformation (Step 3) with or without further purification. For library cloning, the cycle maybe repeated in order to increase the yield of complete plasmids. (B) CPEC cloning of the lacZα gene. The image shows gel electrophoresis analysis of the CPEC reaction product after 1, 2 and 5 cycles (lanes 1–3). 5 µl of the reaction was separated on a 0.8% agarose gel and visualized after ethidium bromide staining. The assembled full-length plasmid was 2644 bp; the empty vector, 2386 bp. A sequence verified, full-length plasmid purified from a bacteria colony was used as a positive control (lane 4). The upper band (2644 bp) represented the relaxed circular form and the fast-migrating lower band, the closed circular form of the plasmid. The molecular weight marker used in this figure was NEB 1 kb DNA ladder (lane M).
Figure 2.
Gene library cloning using CPEC.
(A) Cloning efficiency of CPEC at different cycles using the lacZα codon variants library. (B) Examination of the length of the insert from 64 independent colonies by single-colony PCR. The colonies came from cells transformed with CPEC reaction products after 1, 2, 5, and 15 cycles. The vector-insert ratio in the CPEC reactions was 1∶1. The length of the amplicon with one insert was 592 bp. Single-copy inserts were found in all of the 64 colonies examined. The molecular weight marker used in this figure was NEB 100 bp DNA ladder (lane M).
Figure 3.
Combinatorial gene library cloning using CPEC.
(A) A multi-step strategy combining PCA and CPEC for constructing combinatorial synthetic gene library. As shown in the schematic diagram on the left, in the first step, polymerase cycle assembly (PCA) is used to assemble two sub-libraries into a full-length library; in the second step, CPEC is employed to clone the full-length gp120 gene library inserts (1.7 kb) into the vector (4.7 kb). The gel electrophoresis picture on the right shows the analysis of CPEC progression after 5, 10 and 20 cycles (lanes 1–3). The molecular weight marker used in this figure was NEB 1 kb DNA ladder (lane M). (B) A one-step strategy of constructing combinatorial library using CPEC. The two sub-libraries and the linear vector were mixed in equal concentrations in the CPEC reaction. After 25 cycles, the reaction product was analyzed by 0.8% agarose gel electrophoresis. Arrow marked the 6.4-kb band representing the full-length plasmid. The molecular weight marker used in this figure was NEB 2-log DNA ladder. (C) Gel electrophoresis analysis of inserts amplified from 16 independent colonies from the gp120 library cloned using the one-step CPEC strategy. The molecular weight marker used in this figure was NEB 1 kb DNA ladder.
Figure 4.
Assembly of multi-component pathway using CPEC.
(A) A schematic diagram of the multi-way CPEC. Any two neighboring fragments share an overlapping region with identical Tm. Multiple cycles are usually needed to drive the reaction to completion. The positions of the two nicks (arrow head) in the final completely assembled plasmid may vary depending on the number, lengths, and sequences of the fragments. (B) Gel electrophoresis analysis of the final assembly product after a 20-cycle CPEC. 5 µl of the reaction was separated on a 0.8% agarose gel and visualized after ethidium bromide staining. The full-length plasmid was 8360 bp. (C) Gel electrophoresis analysis of the multi-way CPEC reaction. 5 µl was taken out of the reaction after 2, 5, and 10 cycles and separated on a 0.8% agarose gel (lanes 1, 2 and 3). The starting lengths of the four fragments were 3280, 2959, 2040, and 171 bp, respectively. The 171-bp band was not visible. (D) Restriction mapping of the isolated plasmids derived from the CPEC reaction. Plasmid DNA from five independent colonies (I-V) were digested with BamHI (lane 2, 8.4 kb), BamHI-XhoI (lane 3, 6.6 kb and 1.8 kb), and NdeI (lane 4, 5.4 kb and 3 kb). Purified plasmids not subjected to restriction digestion are shown in lane 1. The molecular weight marker used in this figure was NEB 1 kb DNA ladder.