Molecular Methods in Microbial Ecology

Molecular Methods in Microbial Ecology

Contact Info: Julie HuberLillie 305x7291jhuber@mbl.edu

Schedule: 22 Sept: Introductory Lecture, DNA extraction24 Sept: Run DNA products on gel

Lecture on PCR Prepare PCR reactions

29 Sept: Analyze gels from PCR Lecture on other molecular methods

Readings: Head et al. 1998. Microbial Ecology 35: 1-21.

Day 1

• Introduction to molecular methods in microbial ecology

• Extract DNA from Winogradsky Columns

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Habitat Culturability (%)Seawater 0.001-0.1

Freshwater 0.25Sediments 0.25

Soil 0.3

From Amann et al. 1995 Microbiological Reviews

The Challenge for Microbial Ecology

How do you study something you can’t grow in the lab?

DNA

mRNA

Transcription

The Solution: Molecular Biology

Protein

TranslationRibosome

•Present in all cells- Bacteria, Archaea and Eukaryotes

•Documents of evolutionary history

•Basis of all molecular biological techniques

Head et al. 1998

Head et al. 1998

DNA extraction from Winogradsky Columns

DNA Extraction1. Lyse cell membrane

a. Chemically detergentb. Physically bead beating

2. Pellet cell membrane, proteins and other cell parts while DNA stays in solution

3. Remove other inhibitors from DNA

4. Mix DNA with acid and salt stick to filter

5. Wash filter-bound DNA several times with alcohol

6. Elute DNA off membrane with pH 8, low-salt buffer

Day 2

• Run an electrophoresis gel of the DNA products extracted from your columns

• Learn about PCR

• Set up PCR reactions using the DNA from your extractions and an assortment of primers

Basics of Gel Electrophoresis

• The gel is a matrix (like jello with holes)

• DNA is negatively charged- will run to positive

• Smaller fragments run faster than larger ones

• Gel contains Ethidium Bromide, which binds to DNA and fluoresces when hit with UV light (WEAR GLOVES!!!)

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L RB MC GG AS BP LS

Genomic DNA

The sum total of all DNA from an organism or a community of organisms

What to do

• Mix 10 µl of your DNA with 2 µl loading buffer

• Load in well on gel

• I’ll load the ladder

• Run it

• Take a picture of it

Head et al. 1998

Head et al. 1998

The Star of the Show: SSU rRNA•Everybody has it

•Contains both highly conserved and variable regions

-allows making comparisons between different organisms

over long periods of time (evolutionary history)

•Not laterally transferred between organisms

•HUGE and growing database

Ribosomes

• Make proteins

• rRNA is transcribed from rDNA genes

70S Ribosome

50S subunit

30S subunit

21 different proteins

16S rRNA

31 different proteins

23S rRNA 5S rRNA

SSU rRNA

Universal Tree of Life

BACTERIA

EUKARYA

ARCHAEA

Modified from Norman Pace

BACTERIA

EUKARYA

You Are Here

ARCHAEA

Polymerase Chain Reaction (PCR)

• Rapid, inexpensive and simple way of making millions of copies of a gene starting with very few copies

• Does not require the use of isotopes or toxic chemicals

• It involves preparing the sample DNA and a master mix with primers, followed by detecting reaction products

Every PCR contains:

• A DNA Polymerase (most common, Taq)

• Deoxynucleotide Triphosphates (A, C, T, G)

• Buffer (salt, MgCl2, etc)

• A set of primers, one Forward, one Reverse

• Template DNA

Typical PCR Profile

Temperature Time Action

95ºC 5 minutes DNA Taq polymerase activation

35 cycles of:95ºC54ºC72ºC

1 minute1 minute1 minute

DNA denaturizationPrimer annealingExtension creation

72ºC 10 minutes Final extension created

Slide courtesy of Byron Crump

Things you can optimize

• Temperature and time to activate Taq polymerase

• Temperature and time to allow primer annealing

• Temperature and time for extension

• Concentration of reagents, especially primers, dNTPs, and MgCl2

• Concentration of template DNA

• Number of replication cycles

• Etc…

Beyond 16S

• Identical 16S = Identical Function

• Target functional genes

Luton et al. 2002

16S rDNA mcrA

Primers we are using• 16S rRNA Bacteria

• 16S rRNA Archaea

• mcrA Methanogens – Methyl coenzyme M reductase

• dsrB Sulfate reducers – Dissimilatory bisulfite reductase

Reagent Volume (µl) per reaction # of reactions final volume Sterile H20 22.7 5X PCR buffer 10 dNTPs (8mM) 5 Taq polymerase (5 Units/µl) 0.3 Tube Master mix Target Template Vol F primer Vol R primer Vol

µl µl µl µl

1 38 Sulfate reducers Column DNA 2 dsr1F 5 dsr4R 5

2 38 Methanogens Column DNA 2 ME1 5 ME2 5

3 38 Bacteria Column DNA 2 8F 5 1492R 5

4 38 Archaea Column DNA 2 20F 5 958R 5

5 38 Archaea + control M. jannaschii

2 20F 5 958R 5

6 38 Nothing - control (water) 2 20F 5 958R 5

Day 3

• Examine gels from DNA and PCR

• Learn about more molecular methods in microbial ecology

Class DNA

Nobu Monica Kenly Marshall

10 kb

3 kb

500 bp

Carrie Chrissy Amy Haruka

Some Problems with PCR

• Inhibitors in template DNA

• Amplification bias

• Gene copy number

• Limited by primer design

• Differential denaturation efficiency

• Chimeric PCR products may form

• Contamination w/ non-target DNA

• Potentially low sensitivity and resolution

• General screw-ups

Carrie Marshall Chrissy Kenly

Amy Nobu Haruka Monica

3 kb

500 bp

3 4 2 1

3 4 2 1

3 4 2 1 3 4 2 1 3 4 2 1

3 4 2 1 3 4 2 1 3 4 2 1

3 kb

500 bp

So you have a positive PCR product: Now what?

• Get “community fingerprint” via T-RFLP

• Get “community fingerprint” via DGGE and sequence bands

• Clone and sequence clones

• Go straight into sequencing (massively parallel sequencing, MPS)

B. Crump

B. Crump

B. Crump

What do you DO with sequences?

• Perform a similarity search (database)

• Align the sequences (common ancestry)

• Build a tree (phylogeny and taxonomy)

BLASTBasic Local Alignment Search Tool

http://blast.ncbi.nlm.nih.gov/Blast.cgi

BLASTBasic Local Alignment Search Tool

http://blast.ncbi.nlm.nih.gov/Blast.cgi

Align Sequences and Relatives

Build a Tree (Phylogeny)

Reconstructing evolutionary history and studying the patterns of relationships among organisms

Classification (who is who)

Luton et al. 2002

16S rDNA mcrA

B. Crump

B. Crump

• Built clone libraries from deep-sea rocks

• Compared them to one another and other habitats

Santelli et al. 2008

Santelli et al. 2008

Community Overlap

Santelli et al. 2008

So you have a positive PCR product: Now what?

• Get “community fingerprint” via T-RFLP

• Get “community fingerprint” via DGGE and sequence bands

• Clone and sequence clones

• Go straight into sequencing (massively parallel sequencing, MPS)

Schematic courtesy of B. Crump

MPS Approaches

From Hugenholtz and Tyson 2008

Platform Million base pairs per run

Cost per base (cents)

Average read length (base pairs)

Dye-terminator (ABI 3730xl)

0.07 0.1 700

454-Roche pyrosequencing (GSFLX titanium)

400 0.003 400

Illumina sequencing (GAii)

2,000 0.0007 35

3,000 species?

How many species in 1 L of vent fluid?

3,000 species?

> 36,000 species!

How many species in 1 L of vent fluid?

Now we know who is there:What next?

• Quantify particular groups: FISH or qPCR

Head et al. 1998

Fluorescent In-Situ Hybridization (FISH)

B. Crump

B. Crump

Fluorescent In-Situ Hybridization (FISH)

Schleper et al. 2005

Quantitative (Real Time) PCR

Real time PCR monitors the fluorescence emitted during the reactions as an indicator of

amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

• Detection of “amplification-associated fluorescence” at each cycle during PCR

• No gel-based analysis

• Computer-based analysis

• Compare to internal standards

• Must insure specific binding of probes/dye

Quantitative (Real Time) PCR

Quantitative PCR

Now we know who and how many:What next?

• Metagenomics

• RNA-based methods

• Many many more…

Metagenomics a.k.a., Community Genomics, Environmental Genomics

Does not rely on Primers or Probes (apriori knowledge)!

Image courtesy of John Heidelberg

Metagenomics

Metagenomics

Access genomes of uncultured microbes:Functional PotentialMetabolic Pathways

Horizontal Gene Transfer…

Metagenomics

From the Most “Simple” Microbial Communities…

•Acid Mine Drainage (pH ~0!)

•Jillian Banfield (UC Berkeley)

•Well-studied, defined environment with ~4 dominant members

•Were able to reconstruct almost entire community “metagenome”

•Tyson et al. 2004

… to the potentially most diverse!

•The Sorcerer II Global Ocean Sampling Expedition

•J. Craig Venter Institute “Sequence now, ask questions later”

•Very few genomes reconstructed

•Sequenced 6.3 billion DNA base pairs (Human genome is ~3.2) from top 5 m of ocean

•Discovered more than 6 million genes… and they are only halfway done!

Venter et al. 2004

Most of these methods are “who is there” not “who is active”

• Use RNA

• Link FISH with activity/uptake

DNA

mRNA

Transcription

Protein

TranslationRibosome

Reverse Transcription PCR (RT-PCR)

• Looks at what genes are being expressed in the environment

• Isolate mRNA

• Reverse transcribe mRNA to produce complementary DNA (cDNA)

• Amplify cDNA by PCR

• Analyze genes from environment

RT-PCR

• RNA + Reverse Transcriptase + dNTPs= cDNA

• cDNA + Primers + Taq + dNTPs = gene of interest

• Who is active? What genes are active?

Metatranscriptomics

Access expressed genes of uncultured microbes

(Some) Problems with Molecular Methods

D/RNA extraction Incomplete sampling

Resistance to cell lysis

Storage Enzymatic degradation

PCR Inhibitors in template DNA

Amplification bias

Gene copy number

Fidelity of PCR

Differential denaturation efficiency

Chimeric PCR products

Anytime Contamination w/ non-target DNA

The “best approach?”

• A little bit of everything!

And the list goes on…

• Optical tweezers• Single cell genomics• Meta-proteomics• Microarrays• Flow Cytometry• Nano-SIMS FISH• In-situ PCR and FISH• …