Methods: Molecular Techniques
OEST 740043008043008
IntroductionIntroduction
Molecular Biology – investigates theMolecular Biology investigates the structure and function of biological moleculesmolecules
Fundamental DataNot limited by cultivationNot limited by cultivationRapid diagnosticsM lti l l A l iMulti-level Analysis
From community to single cell
Introduction
Levels of AnalysisDetection of gene or gene product
Functional capacity
Compare gene sequencesIdentityPh l d E l tiPhylogeny and EvolutionAdaptation
Monitor Gene expressionMonitor Gene expressionReponses to environment or mode of growth(i e biofilm phenotype)(i.e. biofilm phenotype)
Shotgun Methodg
Crude method of sequencing or cloningPrimary step does not involve physical p ymap of source clone
unguidedSequence contig www.scq.ubc.caSequence contigmultiple times
PCR
Polymerase Chain ReactionApplicationspp
Directed cloning/sequencingCommunity FingerprintNumber of Gene Copies
Recombinant DNA Clones
Genetic Sequencing16s rRNA
Phylogenetic analysisConserved Regionsg
Cloning and sequencingq g
Variable regionsinformationinformation
Functional gene isequencing
Mutant Type vs. Wild Type
Direct manipulation of genes using molecular cloning and transformation to alter structure and characteristic of genes
Isolation of genesInsertion of genes into transfer vector Transformation
Recombinant DNA technology, geneRecombinant DNA technology, gene splicing, gene modification, etc.Attachment structural developmentAttachment, structural development, QSS
Mutant vs. Wild TypeypLoss of Function experiments
How: gene knockoutHow: gene knockoutWhy: pinpoint phenotypes controlled by genes
Gain of functions increased functionGain of functions – increased functionTracking –
H WT l d ith ‘f i ’How: WT gene replaced with ‘fusion’ geneWhy: Visualize gene modifications
G E iGene Expression How: Reintroduce gene promoter with protein coding region replaced by reporter genecoding region replaced by reporter geneWhy: Where and When data
DGGEDenaturing Gradient Gel Electrophoresis
Large population analysisg p p yFingerprint of genetic diversityComparison allows the visualization of presenceComparison allows the visualization of presence or absence of particular species.
DGGE
2. Clone 4 SATACGGTCAC..
Vector1. Excise4. Sequence
5. Identify
DNA Micro-arrayy
Snap-shot of gene expressionSnap-shot of gene expressionDifferential Gene Express – Biofilm
phenotypephenotypeResistance
DNA Micro-arrayy
Confocal Scanning Laser Microscopy
In-situ analysisIn-situ analysisGreen Fluorescent Protein (GFP)(GFP)
Pro-spatial and temporal resolutionCon-Somewhat limited
In-situ gene expressionFISHRequire molecular probes
Summary
Molecular techniques are invaluable to understanding the structure of biofilmsunderstanding the structure of biofilms on a biogeochemical level.P idi f l i f i hProviding useful information to the questions posed in biofilm research as to who, where, when and how. As well as identifying how biofilmAs well as, identifying how biofilm microbial populations respond to ario s en ironmental ariabilit andvarious environmental variability and
stress.