IB030 Escherichia Coli K-12 (luxABCDEamp). A Tool for Analysis of Bacterial Killing by Complement and Myeloperoxidase Activities

Přírodovědecká fakulta
jaro 2011
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0/0. 1 kr. Ukončení: z.
Vyučující
Janne Atosuo, Ph.D. (přednášející)
Garance
doc. RNDr. Pavel Hyršl, Ph.D.
Ústav experimentální biologie – Biologická sekce – Přírodovědecká fakulta
Kontaktní osoba: doc. RNDr. Pavel Hyršl, Ph.D.
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Osnova
  • J Atosuo, E.-M Lilius Department of Biochemistry, University of Turku, Turku, Finland Backround: Our aim was to construct a tool for analysis of bacterial killing by complement and myeloperoxidase (MPO) activities on a real-time basis. Materials and Methods: We made the recombinant strain of Escherichia coli K-12 by cloning with luxABCDEamp gene expressing bacterioluciferase enzyme. Expression of whole luciferase operon produces cells capable of emitting bioluminescence (BL) without any addition of substrate. BL was measured using multilabel plate readers. Results: We showed that the level of BL was proportional to amount of viable cells by incubating this microbe in wells of a test plate with serum and MPO and measuring the diminishing BL signal in short time intervals and achieving the kinetic curve of killing. BL signal was gained from the number of bacteria ranging from 107 to 102 live bacteria/well representing 99,999% of killing. The killing kinetics was undergoing exponential decay. From the kinetic curves several parameters for complement and MPO activities could be calculated. These parameters include length of a lag-phase where the activation of complement cascade from C1 to C5 occurs, time of 50%, 90%, 99%, etc, of killing and Km and Vmax for MPO. Conclusions: The real-time approach is extremely elegant and provides a valuable tool to researchers whising to elucidate mechanisms of bacterial cell death in response to different antimicrobials. Compared to to conventional plate counting our assay provides more accurate estimation of the bacterial viability with less labor.
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  • Permalink: https://is.muni.cz/predmet/sci/jaro2011/IB030