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TA0100

Sigma-Aldrich

TargeTron Gene Knockout System

Bacterial Gene Knockout

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shipped in

wet ice

storage temp.

−20°C

General description

For detailed information, please visit the TargeTron home page: www.sigma-aldrich.com/targetron.

The TargeTron Gene Knockout System provides optimized reagents and protocols for the rapid and specific disruption of bacterial genes by insertion of group II introns. The method exploits the retrohoming ability of group II introns and utilizes a simple PCR step to re-target the TargeTron group II intron for specific insertion into the host genome. Utility of the technology has been demonstrated for prokaryotic genetic engineering, systems biology and functional genomics approaches.

Compared to existing knockout methodologies, the TargeTron system is quite rapid, simple and robust. The process involves loading the target gene sequence into the well-validated TargeTron algorithm on the Sigma website (www.sigma-aldrich.com/targetronaccess), using one or more of the output primer sets to mutate the TargeTron intron template via PCR, ligation of the fragment into the pACD4K-C linear vector, transformation of the host, induction of gene disruption, selection by kanamycin resistance (insertion of activated marker) and screening for the desired knockout.

Gene knockout using the TargeTron system has been validated in a broad range of bacterial strains such as Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Shigella flexneri, Salmonella typhimurium, and Lactococcus lactis. The system may be modified for use in additional organisms.

Each TargeTron kit provides an access card and password for a specified number of designs (3 EA or 10 EA). The password gives the user access to the extensively validated TargeTron algorithm that predicts optimal intron insertion sites and designs primers for mutating the intron to insert into those sites. After selection of one or more desirable insertion points, the required oligos are ordered to begin laboratory procedures using the kit. Reagents in the 3 EA kit are sufficient for 3 designs, 12 reactions. Reagents in the 10 EA kit are sufficient for 10 designs, 40 reactions.

Application

TargeTron Gene Knockout System has been used to carry out site-directed mutation of genes within human monocyte-derived macrophages. It has also been used to disrupt the nuclease gene, nuc, in Staphylococcus aureus.

Features and Benefits

Targeted and permanent gene disruption
Simple, streamlined protocol; Knockouts in 3 days or less
Minimal screening to isolate mutants
>90% successful targeted insertion
No cell conjugation or specific host factor requirements

Components

System User Manual
TargeTron Access card (password for on-line target site selection)
Intron PCR Template
EBS Universal Primer
LacZ Control Primers
pACD4K-C Linear Vector (20 ng/μl)
JumpStart REDTaq® ReadyMix®
HindIII 20 U/μl
BsrGI 10 U/μl
10× Restriction Enzyme Buffer

Other Notes

To view the TargeTron Gene Knockout System animation, visit sigma.com/targetronanimation.

Legal Information

JumpStart is a trademark of Sigma-Aldrich Co. LLC
REDTaq is a registered trademark of Merck KGaA, Darmstadt, Germany
TargeTron is a trademark of InGex, LLC

Storage Class

10 - Combustible liquids

wgk_germany

WGK 3

flash_point_f

Not applicable

flash_point_c

Not applicable


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M Chelsea Lane et al.
Journal of bacteriology, 191(5), 1382-1392 (2008-12-31)
MR/P fimbriae of uropathogenic Proteus mirabilis undergo invertible element-mediated phase variation whereby an individual bacterium switches between expressing fimbriae (phase ON) and not expressing fimbriae (phase OFF). Under different conditions, the percentage of fimbriate bacteria within a population varies and
Greta R Nielubowicz et al.
Infection and immunity, 76(9), 4222-4231 (2008-07-16)
Proteus mirabilis, a gram-negative bacterium, is a frequent cause of complicated urinary tract infections in those with functional or anatomical abnormalities or those subject to long-term catheterization. To systematically identify surface-exposed antigens as potential vaccine candidates, proteins in the outer
Staphylococcus aureus ?-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.
Herrera A
Biochemistry, 55(17), 2510-2517 (2016)
Sanath Kumar et al.
Archives of microbiology, 193(3), 201-208 (2010-12-25)
A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these
Tao Wang et al.
Frontiers in cellular and infection microbiology, 8, 192-192 (2018-06-26)
Lipid A is an essential basal component of lipopolysaccharide of most Gram-negative bacteria. Inhibitors targeting LpxC, a conserved enzyme in lipid A biosynthesis, are antibiotic candidates against Gram-negative pathogens. Here we report the characterization of the role of lipid A

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