methods: molecular techniquesmcooney/oest740/biofilms_methods_2_ppt.pdf · introduction molecular...

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.