Genetic Transformation in Escherichia coli K12

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13,541

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36-to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCRgenerated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.bacterial genomics ͉ FLP recombinase ͉ FRT sites ͉ Red recombinase T he availability of complete bacterial genome sequences has provided a wealth of information on the molecular structure and organization of a myriad of genes and ORFs whose functions are poorly understood. A systematic mutational analysis of genes in their normal location can provide significant insight into their function. Although a number of general allele replacement methods (1-7) can be used to inactivate bacterial chromosomal genes, these all require creating the gene disruption on a suitable plasmid before recombining it onto the chromosome. In contrast, genes can be directly disrupted in Saccharomyces cerevisiae by transformation with PCR fragments encoding a selectable marker and having only 35 nt of flanking DNA homologous to the chromosome (8). This PCR-mediated gene replacement method has greatly facilitated the generation of specific mutants in the functional analysis of the yeast genome; it relies on the high efficiency of mitotic recombination in yeast (9). Directed disruption of chromosomal genes can also be done in Candida albicans by using similar PCR fragments with 50-to 60-nt homology extensions (10).In contrast to yeast and a few naturally competent bacteria, most bacteria are not readily transformable with linear DNA. One reason Escherichia coli is not so transformable is because of the presence of intracellular exonucleases that degrade linear DNA (11). However, recombination-proficient mutants lacking exonuclease V of the RecBCD recombination complex are transformable with linear DNA (12). Recombination can occur in recB or recC mutants carrying a suppressor (sbcA or sbcB) mutation that activates an alternative recombination pathway; sbcA activates ...

mentioning

confidence: 99%

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

Datsenko

Wanner

2000

13,169

13,541

“…The fact that CsdA is a helix-destabilizing protein and associates with the ribosome at low temperature indicates a specialized role for CsdA in translation at low temperature. To better understand the physiological role of CsdA at low temperature, a csdA deletion mutant was constructed by linear DNA transformation using a recB recC sbcB strain (13). The deletion of csdA was confirmed by Southern blot analysis of the chromosomal DNA (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…E. coli strains SB221 (7), JC7623 (recB recC sbcB) (13), and the corresponding csdA mutant were used in this study.…”

Section: Methodsmentioning

confidence: 99%

Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli.

Jones¹,

Mitta²,

Kim³

et al. 1996

300

256

A 70-kDa protein was specifically induced in Escherichia coli when the culture temperature was shifted from 37 to 15°C. The protein was identified to be the product of the deaD gene (reassigned csdA) encoding a DEAD-box protein. Furthermore, after the shift from 37 to 15°C, CsdA was exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein in cells grown at 15°C. The csdA deletion significantly impaired cell growth and the synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature. Purified CsdA was found to unwind double-stranded RNA in the absence of ATP. Therefore, the requirement for CsdA in derepression of heat-shock protein synthesis is a cold shock-induced function possibly mediated by destabilization of secondary structures previously identified in the rpoH mRNA.Bacterial adaptation to various environmental stresses has been extensively investigated (reviewed in refs. 1-4). Interestingly, it has been demonstrated that Escherichia coli has an adaptive response not only to high temperature by inducing a group of heat-shock proteins but also to low temperature by inducing a group of cold-shock proteins (5, 6). In contrast to heat-shock proteins, which include protein chaperones required for protein folding and peptidases, cold-shock proteins appear to be involved in various cellular functions such as transcription, translation, and DNA recombination (5, 6).Among the cold-shock proteins of E. coli, CspA has been identified as the major cold-shock protein, which is almost exclusively produced at low temperature at a level of 250,000 molecules per cell (5, 7). The three-dimensional structure of CspA consisting of 69 amino acid residues has been determined, which is composed of five antiparallel (3-sheet structures (8,9). CspA binds to single-stranded DNA (8), and its possible function as an RNA chaperone has been speculated (6). In addition to CspA, E. coli contains a large family of CspA-like proteins consisting of CspB, CspC, CspD, and CspE, among which only CspB is a cold-shock protein (10, 11).In the present paper, we report a newly discovered coldshock protein of 70 kDa, which is also almost exclusively produced upon a temperature shift from 37 to 15°C, similar to the induction of CspA. It was found that this newly identified cold-shock protein is exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein at low temperature. This protein was purified and identified to be the product of the gene that has been known as deaD. This gene had been isolated as a multicopy suppressor for a temperature-sensitive mutation located in the gene encoding ribosomal S2 protein and proposed to encode a putative ATP-dependent RNA helicase based on sequence similarities with other known DEAD-box proteins (12). This protein now is assigned CsdA for cold-shock DEAD-box protein A. We found that this protein has a helix-destabilizing activity. Disruption of the gene resulted in a defect in growth a...

“…Later work by others (32) indicated that the failure of chromosomal DNA to transform calcium chloride-treated E. coli in Mandel and Higa's earlier experiments had resulted from exonucleolytic digestion of fragmented chromosomal DNA by a bacterial enzyme. Luckily, the circularity of R-plasmid DNA molecules had avoided this pitfall.…”

Section: Bacterial Transformation By Plasmid Dnamentioning

confidence: 99%

DNA cloning: A personal view after 40 years

Cohen

2013

In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer's laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning.restriction enzyme | pSC101 | EcoRI | genetic engineering | gene cloning