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Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

Journal Article · · BMC Microbiology
 [1];  [2];  [3];  [3];  [4];  [5];  [6];  [6];  [3];  [6];  [6];  [6];  [2];  [2];  [7];  [8];  [7];  [6];  [3]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); DOE/OSTI
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  3. Michigan State Univ., East Lansing, MI (United States). Dept. of Microbiology and Molecular Genetics
  4. Oregon Health and Science University, Portland, OR (United States). Dept. of Molecular Microbiology and Immunology
  5. Oregon Health and Science University, Portland, OR (United States). Dept. of Molecular Microbiology and Immunology; Univ. of Warwick, Coventry (United Kingdom). Leicester Warwick Medical School
  6. Oregon Health and Science University, Portland, OR (United States). Dept. of Molecular Microbiology and Immunology
  7. Baylor Univ., Houston, TX (United States). College of Medicine. Human Genome Sequencing Center
  8. Univ. of Wisconsin, Madison, WI (United States). Medical School. Dept. of Medical Microbiology and Immunology
+ Show Author Affiliations
Background: The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system. Results: The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci. Conclusion: This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.
Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
Grant/Contract Number:
AC52-06NA25396
OSTI ID:
1626487
Journal Information:
BMC Microbiology, Journal Name: BMC Microbiology Journal Issue: 1 Vol. 5; ISSN 1471-2180
Publisher:
BioMed CentralCopyright Statement
Country of Publication:
United States
Language:
English

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