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Molecular Methods in Microbial Ecology Contact Info: Julie Huber Lillie 305 x7291 [email protected] Schedule: 22 Sept: Introductory Lecture, DNA extraction 24 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 ??? ??? ??? ??? ??? ??? ??? ??? The Challenge for Microbial Ecology Habitat Culturability (%) Seawater 0.001-0.1 0.25 0.25 0.3 Freshwater Sediments Soil How do you study something you can’t grow in the lab? From Amann et al. 1995 Microbiological Reviews The Solution: Molecular Biology DNA Transcription mRNA Translation Ribosome Protein •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 Extraction 1. Lyse cell membrane a. Chemically detergent b. 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!!!) - - - - - - - - - - - Genomic DNA L RB MC GG AS BP LS 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 21 different proteins 30S subunit 16S rRNA 31 different proteins 70S Ribosome 50S subunit 23S rRNA 5S rRNA SSU rRNA Universal Tree of Life BACTERIA BACTERIA You Are Here EUKARYA EUKARYA Modified from Norman Pace ARCHAEA 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: 95ºC 54ºC 72ºC 1 minute 1 minute 1 minute DNA denaturization Primer annealing Extension 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 16S rDNA mcrA Luton et al. 2002 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 Sterile H20 22.7 5X PCR buffer 10 dNTPs (8mM) 5 Taq polymerase (5 Units/µl) 0.3 Tube Master mix Target Template µl # of reactions Vol F primer µl final volume Vol R primer µl Vol 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 2 20F 5 958R 5 6 38 Nothing + control M. jannaschii - 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 10 kb 3 kb 500 bp Monica Kenly Marshall 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 Amy Nobu Haruka Monica 3 4 2 1 3 4 2 1 3 kb 500 bp 3 4 2 1 Carrie 3 4 2 1 Marshall Chrissy Kenly 3 kb 500 bp 3 4 2 1 3 4 2 1 3 4 2 1 3 4 2 1 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) BLAST Basic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov/Blast.cgi BLAST Basic 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) 16S rDNA mcrA Luton et al. 2002 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) MPS Approaches Schematic courtesy of B. Crump Platform Million base Cost per Average read pairs per run base (cents) length (base pairs) Dye-terminator (ABI 3730xl) 0.07 0.1 700 454-Roche pyrosequencing (GSFLX titanium) 400 0.003 400 2,000 0.0007 35 Illumina sequencing (GAii) From Hugenholtz and Tyson 2008 How many species in 1 L of vent fluid? 3,000 species? How many species in 1 L of vent fluid? > 36,000 species! 3,000 species? 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 Fluorescent In-Situ Hybridization (FISH) Schleper et al. 2005 B. Crump 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 Quantitative (Real Time) PCR • 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 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 Metagenomics Access genomes of uncultured microbes: Functional Potential Metabolic Pathways Horizontal Gene Transfer … 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! Venter et al. 2004 •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! Most of these methods are “who is there” not “who is active” • Use RNA • Link FISH with activity/uptake DNA Transcription mRNA Translation Ribosome Protein 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 …