The Selection of Recombinant Binary Plasmids Generated by Gateway® LR Cloning in the Escherichia coli Strain C2110
| Autor: | Shuchi Wu, Bingyu Zhao |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
DNA Replication
Genetic Vectors Molecular Sequence Data Replication Origin Bioengineering Molecular cloning Transfection Applied Microbiology and Biotechnology Biochemistry Transformation Genetic Plasmid Escherichia coli Vector (molecular biology) Cloning Molecular Molecular Biology T-DNA Binary system Plasmid preparation Genetics ColE1 Base Sequence biology Protoplasts DNA Restriction Enzymes Sequence Analysis DNA Agrobacterium tumefaciens biology.organism_classification Transformation (genetics) Plasmids Biotechnology |
| Zdroj: | Molecular Biotechnology. 54:125-132 |
| ISSN: | 1559-0305 1073-6085 |
| DOI: | 10.1007/s12033-012-9548-1 |
| Popis: | Gateway(®) cloning is widely used in molecular biology laboratories. Various binary vectors used for Agrobacterium-mediated plant transformation have been modified as destination vectors that are convenient for the sub-cloning of targeted genes from Entry plasmids. However, when the destination and Entry plasmids have the same antibiotic resistance genes for bacterial selection, the non-recombinant Entry plasmid in the LR reaction mixture can compete with the recombinant destination plasmid during bacterial transformation and selection. Methods for the effective selection of recombinant destination plasmids are highly desirable. In this study, we demonstrated that Escherichia coli strain C2110, which is defective in DNA polymerase I (pAL1), could be used to select a recombinant binary destination plasmid with a RK2 replication origin, while the replication of the Entry plasmid with a ColE1 replication origin was inhibited. Plasmid DNA isolated from C2110 by a traditional mini-prep kit was used for restriction enzyme digestion, DNA sequencing, and Arabidopsis protoplast transfection. The binary plasmid in C2110 was also efficiently mobilized into Agrobacterium tumefaciens via the tri-parental conjugation method. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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