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What a Great Time to Teach & Do Research with Undergrads!!

As of 1/3/08: Domain Published Ongoing Archaea 50 68 Bacteria 577 1628 Eucarya 77 891 (+ 114 metagenomes + GEBA plan). What a Great Time to Teach & Do Research with Undergrads!!. So many questions! So many great tools! Too few researchers!. So much data!.

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What a Great Time to Teach & Do Research with Undergrads!!

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  1. As of 1/3/08: DomainPublishedOngoing Archaea 50 68 Bacteria 577 1628 Eucarya 77 891 (+ 114 metagenomes + GEBA plan) What a Great Time to Teach & Do Research with Undergrads!! So many questions! So many great tools! Too few researchers! So much data! *** So much for me & my students to do!!!***

  2. Agrobacterium bv. 2 & 3 strains (NSF grant w/ 7 partners) 2 Xenorhabdus species (USDA grant w/ 6 partners) Ammonifex degensii (JGI pilot program w/ 10 partners) HiramGenomicsInitiative Teaching Research Collaborations Hiram Students High school Students Recruiting Chromohalobacter salexigens (w/Purdue Univ. & DOE-JGI) Sphingomonas elodea (w/Monsanto Co.) Azotobacter vinelandii (NSF grant w/ 4 partners)

  3. Hiram Genomics Initiative Agrobacterium Other Genome Projects Genome Project Sphingomonas Chromohalobacter Xenorhabdus Azotobacter elodeasalexigensbovienii& nematophila vinelandii Functional Genomes of Native Genomics of K84 (bv. 2) Tumor Strain C58 & S4 (bv. 3) Genetic/ Genetic/ Genetic/ Gap Genetic/ Survey (biovar 1) Physical Map Physical Map Physical Map Closure Physical Map (high(Genetics) (Genetics)(Genetics & (Independent (Genetics & schools)high schools) Research) IndependentGap Research) Closure (Independent Sequence Sequence Research) Annotation Annotation (MolCell, Genetics, (Independent & Biochem) Research) Gene Mutant Gap Sequence Disruptions Screens Closure Annotation (MolCell &(MolCell & (Independent (Genetics & Independent Independent Research) Independent Research) Research) Research)

  4. What prevents us from incorporating original research into the lab component of undergraduate courses? Must excite students – move into independent research projects Must excite us Must teach key skills & concepts Must be doable within time, space, & budget constraints Must be successful as measured by the norms of science – effective training for the future, presentations at conferences, & publications Bridging the Teaching-Research Gap Within Undergraduate Courses

  5. 1X Sequencing Coverage Subgenomic Mega-fragments Subgenomic Libraries Overlaps in Small Pieces to Form Contigs 6-8X Sequencing Coverage Genome Genetic/ PhysicalMap Gap Closure Join Large Pieces into Sequenced Genome Random Pieces Annotation of Contig Ends Shotgun Genomic Libraries Annotation Functional Genomics Basics of a Genome Project

  6. 1X Sequencing Coverage Subgenomic Mega-fragments Subgenomic Libraries Overlaps in Small Pieces to Form Contigs 6-8X Sequencing Coverage Genome Genetic/ PhysicalMap Gap Closure Join Large Pieces into Sequenced Genome Random Pieces Annotation of Contig Ends Shotgun Genomic Libraries Annotation Functional Genomics Opportunity Sequence Annotation

  7. 10 kb 0 kb 20 kb Annotation Pipeline • Gene finding & operon prediction • Blast & global sequence alignments • Protein domain prediction • Protein localization prediction • Functional prediction • Functional call, linkage to experimental data, & testable hypotheses (community involvement)

  8. Beyond 1st Pass AnnotationStudents as 2nd Pass Annotators Chromohalobacter salexigens annotation by Biochem students to test the hypothesis that proteins in halophiles are more acidic than their homologs in nonhalophic relatives • PSORT (cellular localization) • BLAST (homologs in E. coli & P. aeruginosa) • MW/pI (pI determination)

  9. Opportunity Testing Bioinformatics Predictions 1X Sequencing Coverage Subgenomic Mega-fragments Subgenomic Libraries Overlaps in Small Pieces to Form Contigs 6-8X Sequencing Coverage Genome Genetic/ PhysicalMap Gap Closure Join Large Pieces into Sequenced Genome Random Pieces Annotation of Contig Ends Shotgun Genomic Libraries Annotation Functional Genomics

  10. Functional Genomics Constructing Gene Disruption Mutants • Pick genes of interest to you and/or genes with putative functions that are testable within your course • Design PCR primers (or have students do so) to amplify an internal portion of a gene • Clone PCR product & confirm by restriction mapping • Introduce cloned PCR product into wildtype and select for single crossover gene disruption gene of interest in A. tumefaciens genome Cbr portion plasmid portion plasmid pCR2.1 of gene of gene cannot replicate in Agrobacterium Cbr

  11. Functional Genomics Constructing Gene Disruption Mutants • 76 genes disrupted since spring of 2002 by MolCell students • 49 genes encoding specific enzymes: multiple genes involved in sucrose metabolism 2 aconitases 4 malate dehydrogenases – only 2 with definable impact 9 penicillin-binding proteins • 27 genes encoding two component systems (mostly response regulators): massive screen of 23 mutants across 54 treatments (covering 12 different environmental variables)

  12. Functional Genomics Example = 2 Aconitases in Agrobacterium C58 wildtype A. tumefaciens from LB plate (pH7) A. tumefaciens acnA- mutant from LB plate (pH7) wt acnA- • One group wanted to look at motility!? • Motility is one process regulated post-transcriptionally by apo-AcnB in E. coli

  13. Functional Genomics Forward Genetics Screens Transposon mutagenesis Mutant screening & characterization Recovery of Tn insertion site Sequence off of Tn end to identify mutated gene

  14. Forward Genetic Screens High School Students Can Do It • Auxotrophs are easy to screen & connect to larger issues of metabolism & nutrition - learn bacterial genetics, mutagenesis, connect genes to enzymes to pathways • If needed, college students physically map insertions - restriction mapping of DNA • obtain sequences at insertion sites - learn DNA sequence analysis, connect genotype to phenotype • Real world = multiple classes since 2002 from 5 area high schools

  15. Forward Genetic Screens Hiram Genomics Academy • Each session lasted 3-5 days • Students generated mutants, screened for phenotypes, recovered Tn insertion sites for sequencing, & learned some bioinformatics • high school students + 11 Hiram students generated & screened 10,500 mutants for 10 different phenotypes, & identified 110 mutants worthy of further study • 79 students from high schools in OH, PA, MI, & IN spread over 5 summer sessions in 2006 & 2007

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