Construction of prokaryotic expression vectors is a core technique in molecular biology and genetic engineering. It refers to inserting the target gene (foreign gene) into a specific vector (circular plasmid) to construct a recombinant vector, so that it can be correspondingly expressed in prokaryotes, usually E. coli.
Primers containing homologous arms and specific sequences are designed first, and the following principles are generally followed:
① The 5' end of the primer contains a 15–25 bp sequence homologous to the vector terminus. For multi-fragment recombination, longer homologous arms (≥30 bp) can significantly increase the correct recombination efficiency;
② The 3' end is the sequence specifically binding to the target gene;
③ The total length ranges from 35 to 75 bp;
The target fragment is then amplified from a cDNA library, genomic DNA or existing plasmids. The yield, specificity and fidelity of the product should be considered during amplification.
The vector needs to be linearized to expose homologous sequences complementary to the ends of PCR products for participation in the recombination reaction. Common methods include single/double restriction enzyme digestion and inverse PCR amplification.
Seamless cloning differs from traditional restriction ligation, which relies on restriction endonucleases to generate sticky ends at specific sequences, followed by covalent ligation of the vector and insert fragments via DNA ligase.
Classical seamless cloning employs the synergistic action of three enzymes (Gibson Assembly): T5 exonuclease, DNA polymerase and DNA ligase;
The 5'→3' digestion activity of T5 exonuclease generates 3' single-stranded overhangs at the double-stranded DNA ends of vectors and fragments. When the vector and fragment share identical sequences in the terminal regions (i.e., homologous arms introduced by primers), their 3' overhangs anneal through base complementary pairing to form a nicked circular intermediate. Subsequently, DNA polymerase fills the single-stranded gaps, and DNA ligase seals the nicks to complete the construction of covalently closed circular recombinant plasmids.
The following principles should be followed when preparing a 10 μL recombination system:
① The total amount of vector and insert fragment ≤ 200 ng;
② The recommended molar ratio of vector to insert fragment is approximately 1:3.
In practical operation, if 50 ng vector (about 5 kb in length) is used, the recommended amount of 1 kb insert fragment is about 30 ng (1:3 molar ratio), which can be adjusted within the range of 20–40 ng according to experimental requirements.
According to the resistance marker of the recombinant vector, the seamless cloning reaction product is added into competent cells. Single colonies containing the recombinant vector are obtained by antibiotic screening, and colony PCR or sequencing is performed to verify the correctness of the inserted fragment.
Recommended operating conditions: Add 5–10 μL recombination reaction product into 50 μL competent cells, incubate on ice for 30 min, perform heat shock at 42°C for 45 s, immediately place on ice for 5 min, then add to 500 μL LB or SOC medium, incubate at 37°C with shaking for 40 min, spread on plates containing the corresponding antibiotics, and incubate at 37°C until single colonies appear. Pick single colonies on the next day for colony PCR (using universal vector primers or gene-specific primers) or send samples for sequencing verification.
Acquisition of Target Fragments
Preparation of Linearized Vector
Seamless Cloning
Transformation and Verification
