Counter‐Selection Method for Markerless Allelic Exchange in Bordetella bronchiseptica Based on sacB Gene From Bacillus subtilis

2020 ◽  
Vol 59 (1) ◽  
Author(s):  
Nicolás Ambrosis ◽  
Julieta Fernández ◽  
Federico Sisti
Keyword(s):  
Bacillus Subtilis ◽  
Selection Method ◽  
Bordetella Bronchiseptica ◽  
Allelic Exchange ◽  
Counter Selection ◽  
Sacb Gene

scholarly journals Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon

1991 ◽  
Vol 5 (6) ◽  
pp. 1447-1457 ◽  
Author(s):  
I. C. Blomfield ◽  
V. Vaughn ◽  
R. F. Rest ◽  
B. I. Eisenstein
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Temperature Sensitive ◽  
Allelic Exchange ◽  
Sacb Gene

Altered fructan accumulation in transgenic Lolium multiflorum plants expressing a Bacillus subtilis sacB gene

2001 ◽  
Vol 20 (3) ◽  
pp. 205-212 ◽  
Author(s):  
X.D. Ye ◽  
X.L. Wu ◽  
H. Zhao ◽  
M. Frehner ◽  
J. Nösberger ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Lolium Multiflorum ◽  
Sacb Gene

Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene

Gene ◽  
1989 ◽  
Vol 78 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Michael F. Hynes ◽  
Jürgen Quandt ◽  
Michael P. O'Connell ◽  
Alfred Pühler
Keyword(s):  
Bacillus Subtilis ◽  
Direct Selection ◽  
Sacb Gene ◽  
Selection For

scholarly journals New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria

BioTechniques ◽  
2021 ◽  
Vol 70 (2) ◽  
pp. 116-119
Author(s):  
Adrian Mejia-Santana ◽  
Cameron J Lloyd ◽  
Karl E Klose
Keyword(s):  
Bacterial Species ◽  
Coli Strain ◽  
Dna Assembly ◽  
Gram Negative Bacteria ◽  
Cloning Vectors ◽  
Quick Method ◽  
Gram Negative ◽  
Allelic Exchange ◽  
Sacb Gene

New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivo assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium.

A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers

2011 ◽  
Vol 57 (5) ◽  
pp. 427-436 ◽  
Author(s):  
Chong Zhang ◽  
Xiaohui Zhang ◽  
Zhengying Yao ◽  
Yaping Lu ◽  
Fengxia Lu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Resistance Gene ◽  
Target Genes ◽  
Genome Structure ◽  
Subtilis Strain ◽  
Gene Target ◽  
Selectable Markers ◽  
Vector Integration ◽  
Bacillus Subtilis 168 ◽  
Counter Selection

This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.

A novel counter-selection method for markerless genetic modification inSynechocystissp. PCC 6803

2012 ◽  
Vol 29 (1) ◽  
pp. 23-30 ◽  
Author(s):  
Yi Ern Cheah ◽  
Stevan C. Albers ◽  
Christie A. M. Peebles
Keyword(s):  
Genetic Modification ◽  
Selection Method ◽  
Counter Selection ◽  
Pcc 6803

scholarly journals Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria.

1996 ◽  
Vol 178 (4) ◽  
pp. 1197-1199 ◽  
Author(s):  
V Pelicic ◽  
J M Reyrat ◽  
B Gicquel
Keyword(s):  
Bacillus Subtilis ◽  
Sacb Gene

scholarly journals Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene.

1993 ◽  
Vol 59 (5) ◽  
pp. 1361-1366 ◽  
Author(s):  
G Recorbet ◽  
C Robert ◽  
A Givaudan ◽  
B Kudla ◽  
P Normand ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Sacb Gene

Biosynthetic specificity of the rhamnosyltransferase gene of Mycobacterium avium serovar 2 as determined by allelic exchange mutagenesis

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3193-3202 ◽  
Author(s):  
Joel N. Maslow ◽  
Vida R. Irani ◽  
Sun-Hwa Lee ◽  
Torsten M. Eckstein ◽  
Julia M. Inamine ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Fluorescent Protein ◽  
Recombinant Expression ◽  
Multicopy Plasmid ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Counter Selection ◽  
Alditol Acetate ◽  
In Trans ◽  
Green Fluorescent

In prior studies, through recombinant expression in Mycobacterium smegmatis, the rtfA gene of Mycobacterium avium was shown to encode a rhamnosyltransferase that catalyses the addition of rhamnose (Rha) to the 6-deoxytalose of serovar 2-specific glycopeptidolipid (GPL). Whether RtfA also catalyses the transfer of Rha to the alaninol of the lipopeptide core is unknown. An isogenic rtfA mutant of M. avium serovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained the katG gene of Mycobacterium bovis and the gene encoding green fluorescent protein (gfp). Overexpression of KatG in M. avium resulted in increased susceptibility to isoniazid, thus providing counter-selection by enriching for clones that had lost plasmid DNA. Plasmid loss was confirmed by screening for gfp-negative clones to select putative allelic exchange mutants. Two exchange mutants were created, confirmed by Southern hybridization, and demonstrated loss of serovar 2-specific GPL by thin-layer chromatography (TLC). Gas chromatography of alditol acetate derivatives revealed the loss of Rha and the terminal 2,3-O-Me-fucose and preservation of 3-O-Me-Rha and 3,4-O-Me-Rha substituents at the terminal alaninol of the lipopeptide core. Complementation of rtfA in trans through an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724. This result shows that rtfA encodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange for M. avium as a tool for future genetic studies involving this species.

Sign in / Sign up

Export Citation Format

Share Document