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NR-19783   Mycobacterium tuberculosis, Strain CDC1551, Knockout Gateway® Clone Set, Recombinant in Escherichia coli, Plate 1
(Clones)
Price: All BEI Resources products are provided
at no cost to registered researchers.
Description: Mycobacterium tuberculosis, Strain CDC1551, Knockout Gateway® Clone Set, Recombinant in Escherichia coli, Plate 1
Organism: Mycobacterium tuberculosis
Biosafety Level: 1
Availability Status: In Stock
Store at: -80°C or colder
Contributor: Pathogen Functional Genomics Resource Center at the J. Craig Venter Institute
Comments:
Quantity limit per order for this item is 1. This item can be ordered twice a year. Orders over this limit will be sent to NIAID for approval before shipment.

The Mycobacterium tuberculosis (M. tuberculosis), Knockout Gateway® clone set consists of 8 plates which contain 641 sequence validated knockout clones from M. tuberculosis, strain CDC1551. Each open reading frame was constructed with a hygromycin selectable gene replacement marker in vector pDEST-YUB, a Gateway® compatible adaptation of the cosmid cloning vector pYUB854 and cloned in Escherichia coli (E. coli) DH10B-T1 cells. The final construct also contains the ß-lactamase gene to confer ampicillin resistance for plasmid selection in E. coli. The sequence was validated by full length sequencing of each clone with greater than 1X coverage and a mutation rate of less than 0.2%.

Information related to the use of Gateway® Clones can be obtained from Invitrogen™. A PCR product representing a functional hygromycin resistance cassette was assembled with chromosomal amplicons of approximately 600 base pairs of the regions flanking each gene targeted for replacement. The three fragments (left flank, hygromycin resistance gene, right flank) were amplified and cloned into pDONR™ entry vectors (Invitrogen™). Recombination was facilitated through an attB substrate (attB-PCR product or a linearized attB expression clone) with an attP substrate (pDONR™ vector) to create an attL-containing entry clone using the three-fragment MultiSite Gateway® Pro method. The hygromycin resistance cassette was sequence verified and experimentally verified through hygromycin resistance of DH10B-T1 E. coli cells. The final destination construct was confirmed by restriction digestion analysis. Please refer to the Invitrogen™ Gateway® Technology Manual for additional Gateway® product details.

Each inoculated well of the 96-well plate contains approximately 60 µL of E. coli culture (strain DH10B-T1) in Luria Bertani (LB) broth containing 100 µg/mL ampicillin supplemented with 15% glycerol.

Production in the 96-well format has increased risk of cross-contamination between adjacent wells. Individual clones should be purified (e.g. single colony isolation and purification using good microbiological practices) and sequence-verified prior to use. BEI Resources does not confirm or validate individual mutants provided by the contributor.
Citations: Acknowledgment for publications should read "The following reagent was obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, Strain CDC1551, Knockout Gateway® Clone Set, Recombinant in Escherichia coli, Plate 1, NR-19783."
Additional Information: Limited Use Label License
NR-19783 JCVI Plate 01 Map
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  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.

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