Molecular Biotechnology Programme Uppsala University School of Engineering
UPTEC X 01 045 Date of issue 2001-12
Author
Carl Otto Öqvist
Title (English)
Analysis of hybrid histidine kinases by gene manipulation in Streptomyces coelicolor
Title (Swedish)
Abstract
Mutants of the Gram-positive bacterium Streptomyces coelicolor were constructed lacking genes encoding two hybrid histidine kinases and the resulting phenotypes were studied. The genes were removed by using a novel recombination system, which uses an engineered strain of E. coli, on cosmids from a genomic cosmid library. Genes on the cosmids were replaced using a linear DNA cassette amplified by PCR. After replacing the genes, the mutant alleles were introduced into Streptomyces coelicolor by transformation and homologous recombination. The inserted antibiotic resistance cassette could be specifically removed from the engineered cosmid using Cre recombinase, leaving an exact deletion of the targeted gene. One of the hybrid histidine kinases, together with a downstream regulator, was required for aerial mycelium formation, which is an important part of the developmental life cycle of this organism.
Keywords
Hybrid histidine kinases, recombination, PCR, Streptomyces coelicolor, Cre recombinase Supervisors
Klas Flärdh
ICM, Uppsala University
Examiner
Gerhart Wagner
ICM, Uppsala University
Project name Sponsors
Language
English
Security
ISSN 1401-2138 Classification
Supplementary bibliographical information Pages
20
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