Positive selection of recombinant DNA by CcdB.
نویسنده
چکیده
منابع مشابه
Construction of T-vector derived from pBluescript ΙΙ SK with a positive selection marker, a rapid system for cloning
A rapid DNA cloning system is a research interest of many scientists. TA cloning is one of the methods used for the cloning of PCR-amplified DNA molecules. The TA cloning method is a convenient and labor-saving replacement to traditional, restriction enzyme-mediated cloning strategies. A T-vector called pBlueskript ΙΙ SK-1 with the lethal gene ccdB was designed to construct a positive selection...
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Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expressio...
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BACKGROUND In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. RESULTS In our case study, t...
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The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactiv...
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ورودعنوان ژورنال:
- BioTechniques
دوره 21 2 شماره
صفحات -
تاریخ انتشار 1996