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Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX
Published on: June 27, 2017
[Construction of directional T vector for gene cloning and expression]
Xing Zhong1, Chao Zhai, Liang Chen
1Faculty of Life Sciences, Hubei University, Wuhan 430062, Hubei, China.
Summary
This study introduces a novel directional T vector, pETG, simplifying gene cloning and expression. This method achieves nearly 100% recombinant efficiency, eliminating complex screening for molecular biology applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Gene Cloning
Background:
- Traditional T vector cloning methods involve difficult recombinant identification and lack directional cloning capabilities.
- These limitations hinder efficient gene cloning and expression in molecular biology research.
Purpose of the Study:
- To develop a directional T vector, pETG, to overcome the limitations of traditional T vectors.
- To improve the efficiency and simplicity of gene cloning and subsequent protein expression.
Main Methods:
- A 7 bp partial LacO sequence was introduced into DNA fragments for gene cloning.
- The modified DNA fragments were ligated into a Bfu I-digested T vector (pETG) to reconstitute a full-length LacO sequence.
- Blue colonies were selected on LB plates with X-gal, followed by restriction enzyme digestion and PCR identification.
Main Results:
- The directional T vector, pETG, demonstrated nearly 100% recombinant efficiency.
- All identified blue colonies contained directionally inserted recombinants, simplifying screening.
- 103 human liver cDNA genes were successfully cloned, and eight selected clones efficiently expressed corresponding proteins.
Conclusions:
- The constructed directional T vector, pETG, is highly effective for gene cloning.
- This vector significantly simplifies the screening process for recombinants.
- pETG is well-suited for high-throughput gene cloning and protein expression studies.
