The Selection of Recombinant Binary Plasmids Generated by Gateway(®) LR Cloning in the Escherichia coli Strain C2110.
Summary of "The Selection of Recombinant Binary Plasmids Generated by Gateway(®) LR Cloning in the Escherichia coli Strain C2110."
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.
Affiliation
Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA, shuchi@vt.edu.
Journal Details
This article was published in the following journal.
Name: Molecular biotechnology
ISSN: 1559-0305
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/22555851
- DOI: http://dx.doi.org/10.1007/s12033-012-9548-1
Medical and Biotech [MESH] Definitions
Cosmids
Plasmids containing at least one cos (cohesive-end site) of PHAGE LAMBDA. They are used as cloning vehicles.
Plasmids
Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.
Enterohemorrhagic Escherichia Coli
Strains of ESCHERICHIA COLI that are a subgroup of SHIGA-TOXIGENIC ESCHERICHIA COLI. They cause non-bloody and bloody DIARRHEA; HEMOLYTIC UREMIC SYNDROME; and hemorrhagic COLITIS. An important member of this subgroup is ESCHERICHIA COLI O157-H7.
Bacteriocin Plasmids
Plasmids encoding bacterial exotoxins (BACTERIOCINS).
Reproductive Techniques
Methods pertaining to the generation of new individuals, including techniques used in selective BREEDING, cloning (CLONING, ORGANISM), and assisted reproduction (REPRODUCTIVE TECHNIQUES, ASSISTED).
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