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Masterthesis: Quantification of bacterial adhesion on surfaces using genome integrated fluorescence markers
For correct and reliable evaluation of bacterial-material interaction (biofilm) it is important to have reliable in situ methods. One of the quantification methods can be the use of genome integrated gene cassettes encoding fluorescence proteins. The student will help to establish this technique.
Bacteria can adhere on many artificial or natural material surfaces and form biofilm. To understand bacterial-material interactions, it is important to have reliable in situ methods for direct biofilm evaluation. The conventional quantification methods are based on counting cells after staining. Many different stains, dyes or markers for the detection of biofilm have thus been developed. These methods often result in loss of bacterial cells due to multistep procedures including washing steps to remove surplus dye. A more straightforward approach is to use genetically engineered strains which emit light directly upon excitation. Consequently, the need of any washing step to remove surplus dye becomes redundant. To achieve such engineered strains, the use of genome integrated gene cassettes encoding fluorescence proteins is one option. Genome integration leads to a stable expression of a gene of interest in comparison to plasmid based gene expression.
Bacteria can adhere on many artificial or natural material surfaces and form biofilm. To understand bacterial-material interactions, it is important to have reliable in situ methods for direct biofilm evaluation. The conventional quantification methods are based on counting cells after staining. Many different stains, dyes or markers for the detection of biofilm have thus been developed. These methods often result in loss of bacterial cells due to multistep procedures including washing steps to remove surplus dye. A more straightforward approach is to use genetically engineered strains which emit light directly upon excitation. Consequently, the need of any washing step to remove surplus dye becomes redundant. To achieve such engineered strains, the use of genome integrated gene cassettes encoding fluorescence proteins is one option. Genome integration leads to a stable expression of a gene of interest in comparison to plasmid based gene expression.
The project aims at 1) introduction of the genes encoding GFP (green fluorescent protein), Ds Red (Discosoma red fluorescent protein), YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein) into the genomes of the standard hospital pathogens _Staphylococcus aureus_ and _Pseudomonas aeruginosa_, respectively; 2) analysis of bacterial adhesion and biofilm formation with the constructed strains qualitatively by fluorescence microscope and quantitatively by fluorescence microplate reader.
The student will work with various biofilm forming pathogens as S. aureus and P. aeruginosa and establish transformation protocols for these strains. He/She will become familiar with the methods for molecular biology (PCR, plasmid preparation, cloning, sequencing, transformation, gel chromatography, etc), biofilm formation (static and (micro-)fluidic cultures, co-cultures, etc), and biofilm assessment (fluorescence microscope, plate reader and others).
The project aims at 1) introduction of the genes encoding GFP (green fluorescent protein), Ds Red (Discosoma red fluorescent protein), YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein) into the genomes of the standard hospital pathogens _Staphylococcus aureus_ and _Pseudomonas aeruginosa_, respectively; 2) analysis of bacterial adhesion and biofilm formation with the constructed strains qualitatively by fluorescence microscope and quantitatively by fluorescence microplate reader. The student will work with various biofilm forming pathogens as S. aureus and P. aeruginosa and establish transformation protocols for these strains. He/She will become familiar with the methods for molecular biology (PCR, plasmid preparation, cloning, sequencing, transformation, gel chromatography, etc), biofilm formation (static and (micro-)fluidic cultures, co-cultures, etc), and biofilm assessment (fluorescence microscope, plate reader and others).
Dr. Beatrice Gutt
Empa - Laboratory for Biointerfaces
Lerchenfeldstrasse 5
9014 St. Gallen
Switzerland
Tel +41 58 765 70 21
beatrice.gutt@empa.ch
Dr. Beatrice Gutt Empa - Laboratory for Biointerfaces Lerchenfeldstrasse 5 9014 St. Gallen Switzerland Tel +41 58 765 70 21 beatrice.gutt@empa.ch