23082011 Bacterial Tools &Amp; Techniques Lecture J. Hays 23.08.11

8/3/2019 23082011 Bacterial Tools &Amp; Techniques Lecture J. Hays 23.08.11

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Bacterial Tools and Techniques

Reporter genes

Omics

MSc Course Infection & ImmunityJohn Hays
http://www.sears.com/shc/s/p_10153_12605_00930534000P?vName=Tools&cName=Tool+Sets&sName=Home+Owner+Tool+Sets


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Reporter Genes

Fluorescent Proteins - GFP

Luminescent Reactions lux / chemiluminescence

- single component

- light excitation

- mult-component (co-factors, substrates)

- biochemical excitation


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Green Fluorescent Protein (GFP)

originally isolated from the bioluminescent jellyfish Aequoria victoria

bioluminescent molecule that shifts blue light (395nm) to (509nm) green light

protein containing 238 amino acids (26.9 kDa),


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1) Infection assays -

adherence / survival assays

2) In vivo gene expression analysis

screening / selection of differential expression within microbial populations

before after

gfp
http://macmcrae.com/wp-content/uploads/2008/08/purplemonster1.jpghttp://macmcrae.com/wp-content/uploads/2008/08/purplemonster1.jpghttp://macmcrae.com/wp-content/uploads/2008/08/purplemonster1.jpghttp://macmcrae.com/wp-content/uploads/2008/08/purplemonster1.jpghttp://macmcrae.com/wp-content/uploads/2008/08/purplemonster1.jpghttp://macmcrae.com/wp-content/uploads/2008/08/purplemonster1.jpg


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1995 - enhanced GFP (EGFP) - mammalian expression

gene modifications -

superfolder GFP

blue (Y66H) GFP

yellow (T203Y) GFP

infrared GFP
http://upload.wikimedia.org/wikipedia/commons/7/7b/FPbeachTsien.jpg


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Differential Fluorescence Induction Analysis of

Streptococcus pneumoniaeIdentifies Genes Involved in Pathogenesis

Marra et al. 2002 Infect Immun March; 70(3) 1422-1433


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Culture and

PCR-sequence promoters

DNase I-

digestion
http://images.google.nl/imgres?imgurl=http://www.paternityexperts.com/img/9700.jpg&imgrefurl=http://www.paternityexperts.com/Why_160.html&usg=__YhykAsvVGuiLoE5_jAvUj73i2Wo=&h=720&w=784&sz=780&hl=nl&start=7&tbnid=pFIQ5ukwl3zL-M:&tbnh=131&tbnw=143&prev=/images%3Fq%3Dpcr%2Bmachine%2B9700%26gbv%3D2%26hl%3Dnl%26sa%3DG


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Found in many organisms - Vibrio fischeri (lux), American firefly (luc), fish, fungi, algae

Biological emission of light as the result of a chemical reaction

Light emitted by action of luciferase enzyme on a long-chain aldehyde (a luciferin)

In bacteria - luciferin is a reduced riboflavin phosphate (FMNH2)

Bioluminescent Luciferase (Lux genes)
http://upload.wikimedia.org/wikipedia/commons/b/bb/Biolumplate.jpg


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Luminescent lux operon


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Visualizing Pneumococcal Infections in the Lungs of Live Mice

Using Bioluminescent Streptococcus pneumoniaeTransformed

with a Novel Gram-Positive luxTransposon

Francis et al. Infect Immun 2001 69(5):3350-3358

In vivo Applications


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Bioluminescent signal corresponds to the number of CFU

The lux construct is highly stable even after several days in vivo

How would this experiment normally be performed in mice?


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(Enhanced) Chemiluminescence (ex vivo)

Horseradish peroxidase enzyme (HRP)

Addition of luminol and hydrogen peroxide (oxidation)

Light produced
http://en.wikipedia.org/wiki/File:Luminol_chemiluminescence_molecular_representation.svghttp://upload.wikimedia.org/wikipedia/en/1/17/Chemoluminescent_reaction.jpg


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Western Blotting

Applications

Southern Blotting


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- omics

The term - omics refers to the comprehensive analysis of biological systems.

Genome / Genomics

the comprehensive analysis of DNA structure and function.

Proteome / Proteomics

the systematic study of proteins

Metabolome / Metabolomics

the study of small-molecule metabolite profiles

Transcriptome / Transcriptomics

the comprehensive analysis of gene expression within a cell


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Genomics

PFGE Pulsed Field gel Electrophoresis

- rare cutter restriction enzyme + genomic DNA

- pulsed field electrophoresis

- create dendrogram

100

90

80

70

60

50

40

30

2 (4)

6 (4)

16 (9) 159 (2)

23 (1)

40 (1)

MLST Mulit-locus Sequence Typing

- sequence 6 7 housekeeping genes

- assign numbers to sequences (1 - >100)

- compare number sequences

(e.g. 1, 2, 3, 4, 5, 6, 7 and 1, 3, 4, 78, 8, 11, 12)

- perform analysis (eburst)


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dTTP*

Sequencing - Chain Termination Method
http://www.google.nl/imgres?imgurl=http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01licohen/seq2.gif&imgrefurl=http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01licohen/sequencing.html&usg=__3brqpHxqNeBbUw6xV5hsg8c_NMQ=&h=218&w=153&sz=16&hl=nl&start=12&tbnid=TD_cx4VipQ2xlM:&tbnh=107&tbnw=75&prev=/images%3Fq%3Dsequencing%2Bgel%26hl%3Dnl%26gbv%3D2%26tbs%3Disch:1&itbs=1


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(Second Generation)

Roche 454

Illumina - Solexa

Applied Biosystems - Solid


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Biotin tagDNA fragmentation

Emulsion PCR (emPCR)

Amplification on bead

+ co-factors / polymerase /primer / packing beads


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(Third Generation) Nanopore sequencing -

True single molecule sequencing

Single Molecule Real Time


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The Human Microbiome Project

Determine whether individuals share a core human microbiome

Understand whether changes in the human microbiome

can be correlated with changes in human health

A human gut microbial gene catalogueestablished by metagenomic sequencing

Nature 464 59-65 2009

3.3 million microbial genes 576.7 gigabases

each individual at least 160 such species (largely shared)


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Transcriptomics (expression profiling)

Microarray

Second generation sequencing
http://images.google.nl/imgres?imgurl=http://www.fastol.com/~renkwitz/affymetrix_chip.gif&imgrefurl=http://www.fastol.com/~renkwitz/microarray_chips.htm&usg=__eO7EfBwhyzUjIU2Pt6AtXM7kZ7Y=&h=318&w=246&sz=38&hl=nl&start=28&tbnid=Hgf3D4sfuODb9M:&tbnh=118&tbnw=91&prev=/images%3Fq%3Dmicroarray%2Bchip%26gbv%3D2%26ndsp%3D18%26hl%3Dnl%26sa%3DN%26start%3D18


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Microarrays


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Proteomics


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Mass Spectrometry - MALDI-TOF

Matrix Assisted Laser Desorption Ionisation - Time of Flight


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Highly Efficient Classification and Identification of

Human Pathogenic Bacteria by MALDI-TOF MS.

Sen-Yung Hsieh et al.

Molecular and Cellular Proteomics.

2008


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Peptide Mass Fingerprinting

Protein samples separated by SDS-PAGE

Chemical modifications performed (e.g. reduce disulfide bridges)

Proteolytic Enzymes (trypsin, chymotrypsin)

Mass spectrometry (MALDI-Tof)

Peak List of peptide masses generated

Compared to databases e.g. Swissprot, Genbank


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Peptide Mass Fingerprinting

Tryptic digest of Human Serum Albumin MALDI_Tof and peptide mapping to sequence
http://upload.wikimedia.org/wikipedia/commons/6/60/PMF_example_Peptide_Mass_Fingerprinting.jpg


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Metabolomics

E. coli 0157(M534)

E. coli K12(BW2952)

Spot matching via - eg Phoretex software


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Reactome Array: Forging a Link Between Metabolome and Genome

Ana Beloqui,1,* Mara-Eugenia Guazzaroni,1,* Florencio Pazos,2 et al.

Science 9 October 2009:

Vol. 326. no. 5950, pp. 252 - 257


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low-nutrient, heavymetalrich, acidicgeothermal pool

organic pollutantcontaminated

seawater

deep-seahypersaline,anoxic lake


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Reporter genes are still widely used in (microbiological) diagnosis and research

Shift towards whole genomic / population analysis -omics

genomics / transcritpomics / proteomics / metabolomics

Novel techniques that generate masses of (useful) data

Bioinformatics tools need to keep pace with technological developments

New developments are facilitating microbiological Point-of-Care diagnostics

Bacterial Tools and Techniques - Conclusions


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Bacterial Tools and Techniques

Reporter genes

Omics
http://www.sears.com/shc/s/p_10153_12605_00930534000P?vName=Tools&cName=Tool+Sets&sName=Home+Owner+Tool+Sets

Documents

23082011 Bacterial Tools &Amp; Techniques Lecture J. Hays 23.08.11