An efficient method for precise gene substitution in the AcMNPV genome by homologous recombination in E. coli

2003 ◽  
Vol 113 (2) ◽  
pp. 95-101 ◽  
Author(s):  
Wuwei Wu ◽  
Jinwen Wang ◽  
Riqiang Deng ◽  
Xunzhang Wang ◽  
XiongLei He ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Efficient Method ◽  
Gene Substitution ◽  
E Coli

scholarly journals A defect in homologous recombination leads to increased translesion synthesisin E. coli

2016 ◽  
Vol 44 (16) ◽  
pp. 7691-7699 ◽  
Author(s):  
Karel Naiman ◽  
Vincent Pagès ◽  
Robert P. Fuchs
Keyword(s):  
Homologous Recombination ◽  
E Coli

scholarly journals Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Kalpana Dulal ◽  
Benjamin Silver ◽  
Hua Zhu
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Bac Clone ◽  
Dna Viruses ◽  
E Coli ◽  
Recombination System ◽  
Capture Method

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

scholarly journals A miniaturized biotyping system for strain discrimination inEscherichia coli

1993 ◽  
Vol 111 (1) ◽  
pp. 81-88
Author(s):  
P. B. Crichton ◽  
J. M. J. Logan ◽  
D. C. Old
Keyword(s):  
Escherichia Coli ◽  
Efficient Method ◽  
High Index ◽  
Inescherichia Coli ◽  
E Coli

SummaryA two-tier miniaturized scheme of eight tests was devised for biotyping strains ofEscherichia coliin microwell plates. Primary biotypes were defined by positive and negative reactions in tests for fermentation of raffinose, sorbose, dulcitol and 2-deoxy-D-ribose and for decarboxylation of ornithine when read after specified periods of incubation; subtypes were identified within primary biotypes according to results in secondary tests for rhamnose fermentation, lysine decarboxylation and motility. The method gave reproducible results on different occasions of testing.Among 100E. colistrains from various sources, 26 of the 32 possible primary biotypes and 56 full biotypes, as defined by results in both primary and secondary tests, were identified, thus demonstrating a high index of strain discrimination (D = 0·98).The scheme is recommended as a simple, reliable, inexpensive and efficient method of differentiating strains ofE. coli.

scholarly journals Integration of a Gold-Specific Whole E. coli Cell Sensing and Adsorption Based on BioBrick

2018 ◽  
Vol 19 (12) ◽  
pp. 3741
Author(s):  
Li Yan ◽  
Peiqing Sun ◽  
Yun Xu ◽  
Shanbo Zhang ◽  
Wei Wei ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Efficient Method ◽  
Visual Detection ◽  
Specific Binding ◽  
Selective Detection ◽  
Biological Detection ◽  
Integration System ◽  
Major Task ◽  
E Coli ◽  
Specific Binding Protein

Detection and recovery of heavy metals from environmental sources is a major task in environmental protection and governance. Based on previous research into cell-based visual detection and biological adsorption, we have developed a novel system combining these two functions by the BioBrick technique. The gold-specific sensory gol regulon was assembled on the gold-chaperone GolB (Gold-specific binding protein), which is responsible for selectively absorbing gold ions, and this led to an integration system with increased probe tolerance for gold. After being incorporated into E. coli, this system featured high-selective detection and recycling of gold ions among multi-metal ions from the environment. It serves as an efficient method for biological detection and recovery of various heavy metals. We have developed modular methods for cell-based detection and adsorption of heavy metals, and these offer a quick and convenient tool for development in this area.

scholarly journals Intra-Molecular Homologous Recombination of Scarless Plasmid

2020 ◽  
Vol 21 (5) ◽  
pp. 1697
Author(s):  
Yaping Liang ◽  
Yu Zhang ◽  
Liangwei Liu
Keyword(s):  
Homologous Recombination ◽  
Efficient Method ◽  
Dna Amplification ◽  
Accurate Method ◽  
Accuracy Rate ◽  
Plasmid Construction ◽  
Plasmid Dnas ◽  
Circular Plasmid

Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5′- and 3′-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.

scholarly journals DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

Cell ◽  
1994 ◽  
Vol 78 (6) ◽  
pp. 1051-1061 ◽  
Author(s):  
Tsuneaki Asai ◽  
David B. Bates ◽  
Tokio Kogoma
Keyword(s):  
Dna Replication ◽  
Homologous Recombination ◽  
E Coli

scholarly journals Assembly of Radically Recoded E. coli Genome Segments

2016 ◽  
Author(s):  
Julie E. Norville ◽  
Cameron L. Gardner ◽  
Eduardo Aponte ◽  
Conor K. Camplisson ◽  
Alexandra Gonzales ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Next Generation ◽  
Selective Marker ◽  
E Coli ◽  
Cassette Exchange ◽  
Large Potential ◽  
Attachment Sites

AbstractThe large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis

2020 ◽  
Vol 171 (5-6) ◽  
pp. 203-210
Author(s):  
Maria Natalia Alonso ◽  
Wladimir Malaga ◽  
Michael Mc Neil ◽  
Mary Jackson ◽  
Maria Isabel Romano ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mycobacterium Avium ◽  
Efficient Method ◽  
Gene Disruption ◽  
Targeted Gene Disruption

scholarly journals Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Mikhail Nefedov ◽  
Lucia Carbone ◽  
Matthew Field ◽  
Jacquie Schein ◽  
Pieter J. de Jong
Keyword(s):  
Homologous Recombination ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Pcr Assay ◽  
New Approach ◽  
E Coli ◽  
Functional Studies ◽  
Kanamycin Cassette ◽  
Orangutan Genome ◽  
Bac Libraries

We have developed a new approach to screen bacterial artificial chromosome (BAC) libraries by recombination selection. To test this method, we constructed an orangutan BAC library using anE. colistrain (DY380) with temperature inducible homologous recombination (HR) capability. We amplified one library segment, induced HR at42∘C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.

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