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TA0100
TargeTron™ Gene Knockout System
Bacterial Gene Knockout
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About This Item
UNSPSC Code:
12352202
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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.
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
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
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
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
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Product No.
Description
Pricing
Storage Class Code
10 - Combustible liquids
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
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Francisella tularensis Schu S4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages.
Lindemann SR
Infection and Immunity, 79(2), 581-594 (2011)
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
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
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)
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What is the Department of Transportation shipping information for this product?
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How stable is the knockout using TargeTron, Product TA0100 after several generations?
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Data in Lactococcus lactis mutants passaged for 80 generations illustrated that 100% of the mutants were stable.
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How can I determine possible targets with the Targetron® algorithim? How do I get my primer designs?
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