Jeff Newman, Lycoming College November 5, 2011 Newman Lab Members - PowerPoint PPT Presentation

Incorporating research into the curriculum: NextGen sequence data in the General Biology (Freshman!) and Molecular Biology laboratories. Jeff Newman, Lycoming College November 5, 2011

Newman Lab Members

Lab Members • Rhizobium etli, E.coli FGARAT – Diana Burley’97, Kathy Roberts’98, Kevin Ferguson’98, Jon Cook’98, Kim Mistiszyn’99, David Wilson’99, Lori Schultz’99, Rachel Lawton’99, Laura Singer’99, Julie Wagner’00 • Human, Staphylococcus aureus FGARAT – Anna Bucher’00, Missy Stokes’01, Melissa Fogg’01, Amy Mayhew’02, Andy Cardillo‘02, Chris Brennan’02, Kristen Skvorak’02, Chris Robbins’03, Stefanie Mensch’03, Christy Boob’04, John Mazzulo’04, Jason Catanzaro’04, Deanne Greene’04, Justin Jay’05, Breann Wolfe’05, Jenny Kinne’05, Denise Greene’05, Marla Yates’05, Jennifer Leader’04, Michael Powell’05, Erica Walsh’05, Bethany Mingle’05, Kim McDowell’06, Andy Lutzkanin’06. • Heterodimer FGARAT & Bioinformatics: Kevin Frederick’01, Mitch Marzo’01, Liz Sehi’06, Jessica Bennett’07, Matt Wright’08 • Other work: Mark McCleland’99, Matthew Georgy’99, Josh Stutzman’00, Tyler Hoffman’12 • Novel Microbes : Will Tumbusch’06, Kellie Cicconi’07, Tyler Marcinko’08, Pat Hayes’09, Brittane Strahan‘09, Allison Batties‘10, Damian Mariano‘10, Samantha McKenna‘10, Alicia Schueck’10, Stephanie Woodhouse‘10, KC Failor‘11, Katherine Smith‘11, Kristen Collins‘11, Karen Kirk-V, Melissa Cashner‘11, Krissy Harstead ’12, Jordan Krebs‘13, Trisha Duncan’13, Logan Mariano ’13, Clark Thompson ‘13 • Color Key: BS-level jobs, Health Care/Vet, Graduate School, Still at Lyco

The Context: Teaching Biology @ Lycoming College • Lycoming College is a National Liberal Arts College with ~1400 students • Standard teaching load is 12 “contact hours” (2 ch for 3 hr lab) – Member of the “Carnegie 44” • No credit for IS or Honors or Chair – Graduate 35-40 Bio majors/year • 50% Pre-health (Anat & Physiol) • Fall (MD,DO,PA,PT,DMD,VMD) – Intro Bio with 2 or 3 labs • 20% Ecology – Upper level with 1 lab (Molecular • 20% General (“Comprehensive”) Biology or Genome Analysis or Research Methods) • 10% Cell & Molecular • Spring – 7 Bio faculty + 1 part time adjunct – Microbiology with 2 labs (2 x 2 hr/wk) – Biochemistry with lab • No Specific Research Funding

My Institution’s Standard Teaching Load (in the sciences) is 1. < 10 real hours/week in class & lab 0% 2. 10-12 real hours/week in class & lab 0% 3. Exactly 12 real hours/week in class & lab 0% 4. 12-15 real hours/week in class & lab 0% 5. >15 real hours/week in class & lab 0%

Is research/scholarly activity required for tenure and/or promotion? 1. We do not have the opportunity to do research 2. Research is NOT required but is encouraged. 3. Modest research activity is required for tenure, publication is required for promotion 4. Publication is required 5. Extramural funding is required 0% 0% 0% 0% 0% 1 2 3 4 5

Do you ID True Unknowns in Micro Lab? A. No, not in any course 0% B. Yes, in Gen Micro but no 16S rDNA 0% C. Yes, in Gen Micro with 16S rDNA seq 0% D. Yes, in Upper Level Micro but no 16S rDNA 0% E. Yes, in Upper Level Micro with 16S rDNA seq 0% 10 Countdown

Solution: Do Research in Class/Lab with students as your research assistants. 10 week Unknown Microbe Lab • Aseptic technique, inoculation • Staining & Microscopy • Temp, O 2 requirements, antibiotics • Carb metabolism/fermentation • Nitrogen/amino acid metab • Differential & Selective media • Bergey’s Manual Lycoming Loyalsock • Biolog GenIII plates Creek Creek • Fatty Acid Methyl Ester (FAME) Analysis • 16S rDNA PCR & sequencing • Search sequences at ________________ • MEGA5 for multiple sequence alignment/ construction of NJ Phylo tree • API test $trip$ with research organisms

If you have a 16S rDNA sequence, what database/tool do you use? A. Don’t know, have never done it. 0% B. BLAST vs non-redundant (nr) GenBank at NCBI 0% C. Classifier at the Ribosomal Database Project (RDP) 0% D. Seq Match at RDP 0% E. Identify at EzTaxon.org 0% F. other 0%

Sequence Analysis • EZTaxon.org – curated database, can search type strains that define species – Species must be cultured, “officially” named & published – <98.5% identity to type strains is indicative of new species • NCBI – contains many more sequences – including those from metagenomic, non- culture based approaches – Little information associated with many sequences, other than source of the sample – Phylogeny of organism can often be determined

Method Background – Sequencing • PCR products from pure cultures and cloned PCR products from uncultured organisms can be sent for sequencing. • We use Agencourt/Beckman/Coulter – Slower (1 -2 week turnaround) but less expensive ($2.50/Rxn) than other companies, higher quality service – they purify PCR samples

Documenting Discoveries of Diversity http://www.lycoming.edu/~newman

Participant Bioinformatics Activity Bacillus subtilis • 16S rRNA sequence analysis in Trichococcus patagoniensis EMW Staphylococcus aureus Microbiology Exiguobacterium undae Lactococcus lactis – One read with EzTaxon Streptococcus pyogenes Corynebacterium callunae – View trace with MEGA Streptomyces coelicolor Oerskovia jenensis – 16S rRNA MSA and tree with Arthrobacter aurescens MEGA5 Prochlorococcus marinus Geovibrio ferrireducens Cytophaga hutchinsonii Chryseobacterium indologenes Blastopirellula marina Helicobacter pylori Bdellovibrio bacteriovorus Neisseria gonorrhoeae Aquaspirillum sinuosum Pseudomonas aeruginosa Escherichia coli Acinetobacter johnsonii Psychrobacter maritimus MC1 Nitrospira moscoviensis Chloroflexus aurantiacus Thermomicrobium roseum Aquifex pyrophilus 0.05

Microbiology Course Feeds into Research • Environmental unknowns cultured & characterized in Microbiology course • Colony PCR of 16S rDNA with primers 27f & 1492r, 1 Sanger sequencing rxn • Compare sequence to validly published type strains (Eztaxon.org) >99.0% identical <99.0% identical Species identification • Fully sequence both strands of nearly complete 16S rDNA - Submit Sequence to GenBank assigned - ClustalW Alignment, Neighbor Joining Tree to infer phylogenetic relationships • Obtain closest relatives as reference strains • Morphological/Metabolic characterization w/standard tests - Colony morphology, color, Gram stain, wet mount - Temperature, O 2 , pH, NaCl requirements Publish new - Carbohydrate & Nitrogen metabolism - Exoenzymes, differential and selective medium species in • API Test Strips (50 CH, 20E/NE, ZYM) IJSEM • Fatty Acid Methyl Ester (FAME) Analysis (MIDI) • Biolog GenIII metabolic profile • Family-specific tests such as Multi Locus Sequence Analysis, pigment analysis, respiratory quinones, polar lipids, cell wall amino acids. • Deposit Strains in Culture Collections (e.g. ATCC, DSMZ, CCUG, JCM, ARS/NRRL)

My exposure to new species descriptions in IJSEM, Taxonomy & Systematics is 1. Never use it, avoid topic in class 0% 2. Occasionally look up original species descriptions 0% 3. Use it all the time, check IJSEM every month 0% 4. Damn splitters & lumpers and keep changing names 0%

Obtain Complete 16S rRNA sequence rRNA1 27f 530f 785f 1114f 16S rRNA gene ~ 1500 bp 1100r 1492r rRNA2 Figure 2. 16S rDNA Sequence Analysis – Top schematic shows the location of PCR and sequencing primers. Lower diagram illustrates the reads assembled into the final consensus sequence.

How are bacterial species defined? • The Gold Standard – Less than 70% DNA-DNA hybridization • % Identity with 16S rDNA sequence – 97% vs. 98.5% • Fig from Stackebrandt & Ebers, 2006, Microbiol. Today 33:152

2 nd Measure of Genomic Uniqueness • DNA-DNA hybridization is “best”, but not archivable • Multi Locus Sequence Analysis with protein coding genes provides better resolution than 16S rDNA – gyrB, rpoB, groEL/hsp60/cpn60, glnA, recA • 95% Average Nucleotide Identity across the genome (ANI)

MLSA Lab in Molecular Biology • Problem: Several “Micrococcus” strains have pigmentation and other traits different from type strains of M.luteus & M.yunnanensis – possibly novel. • All have similar 16S rRNA sequences, what about protein coding genes? Figure M1. Variation in Pigments among Micrococcus strains. M = Micrococcus luteus B-287 T i = Micrococcus sp. AF c = Micrococcus sp. SSX r = Micrococcus sp. TMG o = Micrococcus sp. LYLL c = Micrococcus sp. LYG1 o = Micrococcus sp. LYO1 c = Micrococcus sp. LYE1 c = Micrococcus sp. D7 us = Micrococcus yunnanensis DSM 21948 T

MLSA Lab in Molecular Biology 1. Isolated gDNA from several organisms 2. choose a housekeeping gene, 3. retrieved protein sequence from M.luteus genome site at NCBI, 4. performed BLAST search, 5. choose subset of organisms for multiple sequence alignment 6. Design degenerate primers for conserved sequences, ordered primers 7. Performed/optimized PCR to amplify gene fragment 8. Sequenced PCR product, assembled contig with CAP3 9. Performed multiple sequence alignment 10. Constructed tree and matrix