scholarly journals Adjusting the attB Site in Donor Plasmid Improves the Efficiency of ΦC31 Integrase System

2012 ◽  
Vol 31 (7) ◽  
pp. 1335-1340 ◽  
Author(s):  
Fei Xie ◽  
Qingwen Ma ◽  
Shizhong Jiang ◽  
Zhaorui Ren ◽  
Juan Wang ◽  
...  
Keyword(s):  
Donor Plasmid ◽  
Attb Site ◽  
Φc31 Integrase

Creating transgenic Drosophila by microinjecting the site-specific φC31 integrase mRNA and a transgene-containing donor plasmid

2007 ◽  
Vol 2 (10) ◽  
pp. 2325-2331 ◽  
Author(s):  
Matthew P Fish ◽  
Amy C Groth ◽  
Michele P Calos ◽  
Roel Nusse
Keyword(s):  
Donor Plasmid ◽  
Site Specific ◽  
Φc31 Integrase ◽  
Transgenic Drosophila

scholarly journals UV Light Induces IS10 Transposition in Escherichia coli

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1173-1181
Author(s):  
Zehava Eichenbaum ◽  
Zvi Livneh
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Target Gene ◽  
Uv Light ◽  
Insertion Site ◽  
Donor Plasmid ◽  
Donor And Acceptor ◽  
Low Copy Number ◽  
Uv Induction ◽  
Target Plasmid

Abstract A new mutagenesis assay system based on the phage 434 cI gene carried on a low-copy number plasmid was used to investigate the effect of UV light on intermolecular transposition of IS10. Inactivation of the target gene by IS10 insertion was detected by the expression of the tet gene from the phage 434 PR promoter, followed by Southern blot analysis of plasmids isolated from TetR colonies. UV irradiation of cells harboring the target plasmid and a donor plasmid carrying an IS10 element led to an increase of up to 28-fold in IS10 transposition. Each UV-induced transposition of IS10 was accompanied by fusion of the donor and acceptor plasmid into a cointegrate structure, due to coupled homologous recombination at the insertion site, similar to the situation in spontaneous IS10 transposition. UV radiation also induced transposition of IS10 from the chromosome to the target plasmid, leading almost exclusively to the integration of the target plasmid into the chromosome. UV induction of IS10 transposition did not depend on the umuC and uvrA gene product, but it was not observed in lexA3 and ΔrecA strains, indicating that the SOS stress response is involved in regulating UV-induced transposition. IS10 transposition, known to increase the fitness of Escherichia coli, may have been recruited under the SOS response to assist in increasing cell survival under hostile environmental conditions. To our knowledge, this is the first report on the induction of transposition by a DNA-damaging agent and the SOS stress response in bacteria.

scholarly journals Creation of Golden Gate constructs for gene doctoring

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Nicholas M. Thomson ◽  
Chuanzhen Zhang ◽  
Eleftheria Trampari ◽  
Mark J. Pallen
Keyword(s):  
Free Choice ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Dna Fragments ◽  
Golden Gate ◽  
Donor Plasmid ◽  
Recombination System ◽  
Green Fluorescent ◽  
Λ Red

Abstract Background Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps. Results We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. Conclusions Our plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families.

ΦC31 integrase interacts with TTRAP and inhibits NFκB activation

2009 ◽  
Vol 37 (6) ◽  
pp. 2809-2816 ◽  
Author(s):  
Bing-yin Wang ◽  
Guan-lan Xu ◽  
Cai-hong Zhou ◽  
Ling Tian ◽  
Jing-lun Xue ◽  
...  
Keyword(s):  
Φc31 Integrase

scholarly journals Optimization of Streptomyces bacteriophage φC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells

2009 ◽  
Vol 41 (12) ◽  
pp. 919 ◽  
Author(s):  
Manish Kumar Aneja ◽  
Johannes Geiger ◽  
Rabea Imker ◽  
Senta Üzgün ◽  
Michael Kormann ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Type Ii Cells ◽  
Type Ii ◽  
Φc31 Integrase

A Profile of Native Integration Sites Used by φC31 Integrase in the Bovine Genome

2012 ◽  
Vol 39 (5) ◽  
pp. 217-224 ◽  
Author(s):  
Lijuan Qu ◽  
Qingwen Ma ◽  
Zaiwei Zhou ◽  
Haiyan Ma ◽  
Ying Huang ◽  
...  
Keyword(s):  
Bovine Genome ◽  
Φc31 Integrase ◽  
Integration Sites

Insertion-Duplication Mutagenesis inStreptococcus pneumoniae: Targeting Fragment Length Is a Critical Parameter in Use as a Random Insertion Tool

1998 ◽  
Vol 64 (12) ◽  
pp. 4796-4802 ◽  
Author(s):  
Myeong S. Lee ◽  
Chaok Seok ◽  
Donald A. Morrison
Keyword(s):  
Gene Knockout ◽  
Genomic Analysis ◽  
Combinatorial Theory ◽  
Donor Plasmid ◽  
Uniform Size ◽  
Knock Out ◽  
Duplication Events ◽  
Simplifying Assumptions ◽  
Insertion Tool ◽  
Random Insertion

ABSTRACT To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.

Preliminary study on the DNA-binding properties of phage ΦC31 integrase

Gene ◽  
2011 ◽  
Vol 484 (1-2) ◽  
pp. 47-51 ◽  
Author(s):  
Zhihui Li ◽  
Yuxiang Fang ◽  
Rencheng Wang ◽  
Jinglun Xue ◽  
Jinzhong Chen
Keyword(s):  
Dna Binding ◽  
Binding Properties ◽  
Φc31 Integrase ◽  
Preliminary Study ◽  
Dna Binding Properties

scholarly journals Response to Comment on “RNA-guided DNA insertion with CRISPR-associated transposases”

Science ◽  
2020 ◽  
Vol 368 (6495) ◽  
pp. eabb2920 ◽  
Author(s):  
Jonathan Strecker ◽  
Alim Ladha ◽  
Kira S. Makarova ◽  
Eugene V. Koonin ◽  
Feng Zhang
Keyword(s):  
Escherichia Coli ◽  
Donor Plasmid ◽  
Dna Insertion ◽  
Simple Integration

Rice et al. suggest that the CRISPR-associated transposase ShCAST system could lead to additional insertion products beyond simple integration of the donor. We clarify the outcomes of ShCAST-mediated insertions in Escherichia coli, which consist of both simple insertions and integration of the donor plasmid. This latter outcome can be avoided by use of a 5′ nicked DNA donor.

scholarly journals High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

2014 ◽  
Vol 80 (13) ◽  
pp. 3826-3834 ◽  
Author(s):  
Junjie Yang ◽  
Bingbing Sun ◽  
He Huang ◽  
Yu Jiang ◽  
Liuyang Diao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Modification ◽  
Target Gene ◽  
Donor Plasmid ◽  
Content Type ◽  
Recognition Sites ◽  
Genetic Modifications ◽  
Pcr Targeting ◽  
Λ Red ◽  
Red Recombination

ABSTRACTGenetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of theEscherichia colichromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used forcadAknockout,gdhAknock-in, seamless deletion ofpepD, site-directed mutagenesis of the essentialmetKgene, and replacement ofmetKwith theRickettsiaS-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.

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