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Product Name:
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Borrelia burgdorferi, Signature-Tagged Mutagenesis Library Clone T07TC477 (Gene BB_J48)
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Manufacturer:
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BEI Resources
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Taxonomy:
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Bacteria Classification: Spirochaetaceae, Borrelia
Species: Borrelia burgdorferi
Strain: B31, clone 5A18NP1
Signature-Tagged Mutagenesis Library Clone: T07TC477
Replicon: Linear plasmid lp38
Gene: BB_J48 (conserved hypothetical protein)
Insertion Site1,2: 36257
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Additional Information:
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B. burgdorferi is a Gram-negative, motile spirochete.3 It is a zoonotic, vector-borne pathogen transmitted by ticks and the etiologic agent of Lyme disease, now the most common tick-transmitted disease in the United States.4 B. burgdorferi is predominant in North America, but also exists in Europe.
B. burgdorferi, strain B31 was originally isolated in 1981 from adult ticks (Ixodes dammini) collected from lower vegetation on Shelter Island, New York, USA.3,4 Strain B31 is composed of a 910 kilobase (kb) linear chromosome, 9 circular plasmids (cp) and 12 linear plasmids (lp). Plasmids range in size from 5 kb to 56 kb and total 610 kb.2,5 Continuous passage of B. burgdorferi is known to cause spontaneous loss of plasmids. The complete genome of B. burgdorferi, strain B31 has been sequenced (GenBank: AE000783).6
B. burgdorferi, strain B31, clone 5A18NP1 was derived from the low-passage clone 5A18 of strain B31.7 Clone 5A18NP1 lacks Ip56 and Ip28-4 and the BB_E02 gene (a putative restriction-modification gene on Ip25) was disrupted by homologous recombination resulting in kanamycin resistance.8 Inactivation of BB_E02 results in increased transformation efficiency and therefore, clone 5A18NP1, was used to create the STM library through the mariner-based transposition suicide Himar1 delivery vector, pMarGent, containing aacC1 which confers gentamicin resistance.1,2,9 STM is a negative selection method that identifies clones by unique DNA sequences that are incorporated into the transposable element.2
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Material Provided:
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Each vial contains approximately 0.5 mL of bacterial culture in Revised Barbour-Stoenner-Kelly medium supplemented with 200 µg/mL kanamycin, 40 µg/mL gentamicin and 15% glycerol.
Note: If homogeneity is required for your intended use, please purify prior to initiating work.
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Packing/Storage:
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NR-59645 was packaged aseptically in cryovials. The product is provided frozen and should be stored at -80°C or colder immediately upon arrival. For long-term storage, the vapor phase of a liquid nitrogen freezer is recommended. Freeze-thaw cycles should be avoided.
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Growth Conditions:
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Media:
Revised Barbour-Stoenner-Kelly broth (see Appendix I) with 200 µg/mL kanamycin and 40 µg/mL gentamicin
Revised Barbour-Stoenner-Kelly agar (see Appendix I) with 200 µg/mL kanamycin, 40 µg/mL gentamicin and 0.8% agar
Incubation:
Temperature: 32°C to 34°C (growth at 37°C may result in plasmid loss)1
Atmosphere: Microaerophilic (slower growth occurs under aerobic conditions)1
Propagation:
1. Keep vial in dry ice during inoculations.
2. Inoculate new cultures from scraping of frozen stock into a single tube of Revised Barbour-Stoenner-Kelly Medium.
3. Incubate the tube at 32 to 34°C for 2 to 14 days. Do not shake culture during growth.
Note: Subculturing should be minimized to avoid plasmid loss.1,7
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Disclaimers:
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You are authorized to use this product for research use only. It is not intended for human use. Use of this product is subject to the terms and conditions of the BEI Resources Material Transfer Agreement (MTA). The MTA is available on our Web site at www.beiresources.org. While BEI Resources uses reasonable efforts to include accurate and up-to-date information on this product sheet, neither ATCC® nor the U.S. Government makes any warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. Neither ATCC® nor the U.S. Government warrants that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, use and disposal. ATCC® and the U.S. Government are not liable for any damages or injuries arising from receipt and/ or use of this product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, the U.S. Government, ATCC®, their suppliers and contributors to BEI Resources are not liable for damages arising from the misidentification or misrepresentation of products. |
References:
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1. Norris, S. J., Personal Communication.
2. Lin, T., et al. “Analysis of an Ordered, Comprehensive STM Mutant Library in Infectious Borrelia burgdorferi: Insights into the Genes Required for Mouse Infectivity.” PLoS One 7 (2012): e47532. PubMed: 23133514.
3. Burgdorfer, W., et al. “Lyme Disease – A Tick-Borne Spirochetosis?” Science 216 (1982): 1317-1319. PubMed: 7043737.
4. Johnson, R. C., et al. “Borrelia burgdorferi sp. nov.: Etiologic Agent of Lyme Disease.” Int. J. Syst. Bacteriol. 34 (1984): 496-497.
5. Casjens, S., et al. “A Bacterial Genome in Flux: The Twelve Linear and Nine Circular Extrachromosomal DNAs in an Infectious Isolate of the Lyme Disease Spirochete Borrelia burgdorferi.” Mol. Microbiol. 35 (2000): 490-516. PubMed: 10672174.
6. Fraser, C. M., et al. “Genomic Sequence of a Lyme Disease Spirochaete, Borrelia burgdorferi.” Nature 390 (1997): 580-586. PubMed: 9403685.
7. Purser, J. E. and S. J. Norris. “Correlation Between Plasmid Content and Infectivity in Borrelia burgdorferi.” Proc. Natl. Acad. Sci. USA 97 (2000): 13865-13870. PubMed: 11106398.
8. Kawabata, H., S. J. Norris and H. Watanabe. “BBE02 Disruption Mutants of Borrelia burgdorferi B31 Have a Highly Transformable, Infectious Phenotype.” Infect. Immun. 72 (2004): 7147-7154. PubMed: 15557639.
9. Botkin, D. J., et al. “Identification of Potential Virulence Determinants by Himar1 Transposition of Infectious Borrelia burgdorferi B31.” Infect. Immun. 74 (2006): 6690-6699. PubMed: 17015459.
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Citation:
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Acknowledgment for publications should read “The following reagent was obtained through BEI Resources, NIAID, NIH: Borrelia burgdorferi, Signature-Tagged Mutagenesis Library Clone T07TC477 (Gene BB_J48), NR-59645.”
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Biosafety Level:
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2
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories (BMBL). Current Edition. Washington, DC: U.S. Government Printing Office.
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