Electrotransformation of the Spirochete Borrelia burgdorferi

Samuels, D. Scott

doi:10.1385/0-89603-310-4:253

Cited by 251 publications

(282 citation statements)

References 18 publications

(28 reference statements)

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“…In order to overexpress CsrA Bb in B. burgdorferi, P flgB -csrA Bb -His tag was cloned into the borrelial shuttle vector pBSV2 using restriction enzymes that flank P flgB -csrA-His tag in pCR2.1. The plasmid designated pES25 was used to transform a clonal isolate of B. burgdorferi strain B31 (ML23; lp25 negative) as described previously (51,72). The transformants were selected on BSK-II agar overlay supplemented with kanamycin (200 g/ml) and checked for the presence of pBSV2.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

et al. 2009

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Section: Methodsmentioning

confidence: 99%

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

et al. 2009

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“…B. burgdorferi strain B31 clones A34 (16) and B312 (26) were grown to mid-log phase (ϳ5 ϫ 10 7 cells/ml). Spirochetes were enumerated using dark-field microscopy and a Petroff-Hausser counting chamber and prepared for electroporation as described previously (27). Transformed B31-A34 and B312 cells were selected using gentamicin at a concentration of 40 g/ml.…”

Section: Methodsmentioning

confidence: 99%

“…pBH-lacZEc and pBHflaBp-lacZEc were transformed into electrocompetent B. burgdorferi cells (27). Spirochetes were allowed to recover overnight in BSK II medium without antibiotic selection and then plated in solid BSK medium supplemented with gentamicin.…”

Section: Methodsmentioning

confidence: 99%

“…The intergenic region between guaA and ospC, which includes the ospC promoter and regulatory operator sequences up to the ospC start codon (12,20,21,(37)(38)(39), was amplified from B. burgdorferi genomic DNA with primers 13 and 14 (Table 2), cloned into pCR2.1, sequenced, and digested with BamHI and XhoI for ligation into pBH-lacZBb, creating pBHospCp-lacZBb. Constructs were transformed into electrocompetent B. burgdorferi (27) and ⌬lac E. coli cells.…”

Section: Methodsmentioning

confidence: 99%

“…Within the last 2 decades, the complete genomic sequence of B. burgdorferi strain B31 was published (10,14) and techniques for basic genetic manipulation of B. burgdorferi became available (5,11,13,(27)(28)(29)36). A chloramphenicol acetyltransferase (CAT) gene was the first reporter gene that was fused to B. burgdorferi promoters for analysis of promoter strength (33).…”

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lacZ Reporter System for Use in Borrelia burgdorferi

Hayes

Jewett

Rosa

2010

Appl Environ Microbiol

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Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable ␤-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, ␤-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.

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Laboratory Maintenance of Borrelia burgdorferi

Zückert

2007

CP Microbiology

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This unit describes the methods for growth and storage of Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. It provides the recipe of the standard culture medium, Barbour-Stoenner-Kelly-II (BSK-II) and details its use in broth and solid culture. Methods for short and long-term storage of Borrelia cells are also provided. CAUTION: B. burgdorferi is considered a Biosafety Level 2 (BSL-2) category organism. Work should be performed in a BSL-2 biosafety hood. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information. The use of a biosafety hood also further protects cultures from potential contaminants, which will readily outgrow the spirochetes in the rich culture medium (see Commentary). STRATEGIC PLANNING Borrelia burgdorferi characteristicsBorrelia burgdorferi are slender, highly motile spirochetes 0.2-to 0.5-µm in diameter and up to 30 µm in length. They can be visualized by phase-contrast or dark-field microscopy (UNIT 2A.1). B. burgdorferi stain weakly Gram negative; other stains such as Giemsa or Wright are more appropriate. B. burgdorferi are motile in both low-and high-viscosity media due to periplasmic bundles of seven to eleven flagella attached at each end of the cell cylinder. Intriguingly, these flagella also determine the spirochete's flat-wave cell shape (Motaleb et al., 2000). Opaque white colonies form within or on the surface of solid medium (see Basic Protocol 3). In general, colonies are regularly shaped, up to 3 mm in diameter, with a dense center and a diffuse border. However, colony size and morphology can vary from strain to strain or even within clones of the same isolate (Elias et al., 2002). Borrelia sensitivity to detergentsThe use of disposable labware is recommended since Borrelia cells are sensitive to detergents, residues of which might be present on reusable plastic and glass labware. All reusable labware, such as beakers for medium preparation, should be rinsed with double-distilled water before use. Stock solutionsThe formulation for the standard Borrelia growth medium BSK-II, provided in Reagents and Solutions, follows the recipe described by Barbour in 1984. The medium is made in two steps: an incomplete or basal medium, which can be stored indefinitely at 4 • C or lower, and a complete medium, which is stored up to 2 to 3 weeks at 4 • C. In addition to the basal BSK-II medium, gelatin or agarose stock solutions for liquid or solid BSK-II, respectively, are prepared beforehand and stored indefinitely at room temperature. Rabbit serum is aliquoted in ready-to-use volumes and stored indefinitely at −20 • C or lower. CMRL-1066CMRL-1066 without L-glutamine is available as liquid or powder from a limited number of companies, e.g., Invitrogen (1× solution), or U.S. Biological (powder). The 10× solutions previously sold by different suppliers (e.g., Invitrogen and Sigma) have been discontinued. In powder form, CMRL-1066 should be stored protected from light and humidity (e.g., in a dessicator) at room temperature. Solutions should be stored protected f...

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Electrotransformation of the Spirochete Borrelia burgdorferi

Cited by 251 publications

References 18 publications

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

lacZ Reporter System for Use in Borrelia burgdorferi

Laboratory Maintenance of Borrelia burgdorferi

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