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Bacterial Tools and Techniques
Reporter genes
Omics
MSc Course Infection & ImmunityJohn Hays
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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.jpg8/3/2019 23082011 Bacterial Tools &Amp; Techniques Lecture J. Hays 23.08.11
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1995 - enhanced GFP (EGFP) - mammalian expression
gene modifications -
superfolder GFP
blue (Y66H) GFP
yellow (T203Y) GFP
infrared GFP
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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
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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)
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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
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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=18/3/2019 23082011 Bacterial Tools &Amp; Techniques Lecture J. Hays 23.08.11
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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
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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
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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