Development of system for visualizing natural transformation in real time
Lokhman, Elena (2014)
2014
Master's Degree Programme in Science and Bioengineering
Luonnontieteiden tiedekunta - Faculty of Natural Sciences
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Hyväksymispäivämäärä
2014-06-04
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201406061297
https://urn.fi/URN:NBN:fi:tty-201406061297
Tiivistelmä
Horizontal gene transfer (HGT) is a common event in nature and one of the major sources of novelties and diversity in microorganisms. Natural transformation is one of the HGT options and is the state during which bacteria is capable of taking free genetic material from the environment and recombining it in own chromosome.
Conventional laboratory method for observation of natural transformation and HGT based on growth selection has substantial limitations, such as underestimation of real transformation frequency, use of antibiotic resistance markers and general laboriousness. Therefore, some attempts to visualize HGT directly have been made with employment of fluorescent proteins and luminescence production as an alternative to conventional markers.
This study has made an attempt to create the new system for tracking the natural transformation in the real time with use of luminescence production as a fast measure of transformation extent. Acinetobacter baylyi ADP1 known for its natural competence and easy handling, full genome sequence of which is available, was chosen as a model organism for these manipulations.
The developed system was tested with several combinations of knock-out genes and antibiotic resistance markers, demonstrating the significant divergence in function of each particular combination and revealing additional factors influencing the natural transformation. Therefore, the initial goal to correlate the light production with the amount of positive transformants quantitatively and calibrate the system for more or less universal application proved to be difficult.
Nevertheless, the developed system was successfully applied for trial experiments to study the HGT between A. baylyi ADP1 and Escherichia coli. Additionally, it demonstrated the promising results in observing and confirming two-component simultaneous transformation in real time with potential to be further developed for refining of this experiment. Furthermore, some modifications to the system have been suggested to improve its functioning and further application ideas were proposed.
Conventional laboratory method for observation of natural transformation and HGT based on growth selection has substantial limitations, such as underestimation of real transformation frequency, use of antibiotic resistance markers and general laboriousness. Therefore, some attempts to visualize HGT directly have been made with employment of fluorescent proteins and luminescence production as an alternative to conventional markers.
This study has made an attempt to create the new system for tracking the natural transformation in the real time with use of luminescence production as a fast measure of transformation extent. Acinetobacter baylyi ADP1 known for its natural competence and easy handling, full genome sequence of which is available, was chosen as a model organism for these manipulations.
The developed system was tested with several combinations of knock-out genes and antibiotic resistance markers, demonstrating the significant divergence in function of each particular combination and revealing additional factors influencing the natural transformation. Therefore, the initial goal to correlate the light production with the amount of positive transformants quantitatively and calibrate the system for more or less universal application proved to be difficult.
Nevertheless, the developed system was successfully applied for trial experiments to study the HGT between A. baylyi ADP1 and Escherichia coli. Additionally, it demonstrated the promising results in observing and confirming two-component simultaneous transformation in real time with potential to be further developed for refining of this experiment. Furthermore, some modifications to the system have been suggested to improve its functioning and further application ideas were proposed.