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

Faculty of Science
Spring 2011
Extent and Intensity
0/0. 1 credit(s). Type of Completion: z (credit).
Teacher(s)
Janne Atosuo, Ph.D. (lecturer)
Guaranteed by
doc. RNDr. Pavel Hyršl, Ph.D.
Department of Experimental Biology – Biology Section – Faculty of Science
Contact Person: doc. RNDr. Pavel Hyršl, Ph.D.
Course Enrolment Limitations
The course is offered to students of any study field.
Syllabus
  • 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.
Language of instruction
English
Further Comments
The course is taught only once.
The course is taught: in blocks.
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