Bridging the Teaching-Research Gap in Undergraduate Courses

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

2. 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)

3. 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

4. Example of Success: Agrobacterium Genome Project bacterium hormones DNA food plant cell • Has involved >300 students within course research projects as well as in independent projects (at Hiram College & University of Richmond) since 1996 • 19 student authors on publications in Journal of Bacteriology & Science • >50 student authors on >30 posters presented at research conferences • Successful involvement in collaborations with companies & larger universities

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 Example #1 Generating Combined Genetic/Physical Map

7. rich medium minimal medium Combined Genetic/Physical Map Transposon mutagenesis Mutant screening (auxotrophs?) & characterization Recovery of Tn insertion site Physical mapping (PFGE)

8. CombinedGenetic &PhysicalMaps(J. Bact.181:5160-6)

9. Combined Genetic/Physical MapConnecting Sequence Contigs to Map (Tn5-RL27) 1 2 3 4 1: Digestion with SacII … dilute ligation 2: Transform into pir+ E. coli 3: Sequence off Tn ends … query contigs 4: Contig can be placed on map

10. 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 Example #2 Bioinformatics-based Gap Closure

11. Design PCR primers (one reading off each end) & use them to amplify the missing gap sequence Bioinformatics-based Gap ClosureComparing the Ends of Contigs Gene X? Partner A Contig Partner B Contig BLAST analysis of the right end of contig A reveals the first part of gene X BLAST analysis of the left end of contig B reveals the last part of gene X

12. Bioinformatics-based Gap ClosureExamples from Sphingomonas elodea Partner A ContigPutative JoinPartner B Contig Left end of C452 Glucokinase ORF (gap is a few Right end of C466 reading out bases near codon for AA#71) reading in Right end of C491 cobW ORF (gap is bases Right end of C448 reading out encoding AA#120-500) reading in Right end of C528 g-glutamyl-P reductase ORF (gap Right end of C523 reading out is bases encoding AA#230-235) reading in Left end of C502 Ribonuclease R ORF(gap is bases Right end of C482 reading out encoding AA#420-430) reading in   

13. 500 bp 500 bp 500 500 500 500 Bioinformatics-based Gap ClosureUsing One Genome to Close Another Query Contig from A. tumefaciens C58 Subject Contig 1 from A. vitis S4 Subject Contig 2 from A. vitis S4 ParaGap, a program written by Adam Ewing (Hiram ‘05), uses BLAST analysis between contigs of two related genomes to find areas of synteny (shared gene order) that can be used to orient contigs with respect to each other

14. 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 Example #3 Sequence Annotation

15. 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)

16. BeyondFirstPass AnnotationStudents as Pathway Experts Genetics students assigned a pathway to compare 2 strains of Agrobacterium in terms of gene content, gene order, etc. L-Histidine • There are 9 enzymes involved in the histidine biosynthesis pathway and all the enzymes have one subunit type each. HisD, also called histidinol dehydrogenase, functions twice in the pathway accepting both L-histidinol and L-histidinal as substrates. • There are no genes missing for this biosynthetic pathway in either the C58 or the S4 genome.

17. BeyondFirstPass Annotation L-Histidine • There is gene redundancy for hisC, with 2 copies in C58 and 4 copies in S4. The two genomes share one copy (Atu1011/Avi1423) that is on ChrI in both genomes. The two genomes share another copy that is on ChrII in C58 (Atu3612) but still on ChrI in S4 (Avi4034). Both of these shared copies are ancestral throughout the Rhizobiaceae. Then there are 2 more hisC genes in S4. One of these is on ChrI (Avi2955) and appears to be an ancestral 3rd copy that was lost sometime in biovar 1. The other gene is found on the 130kb plasmid (Avi9607) and has closest extant homologs in Ralstonia and Pseudomonas.

18. BeyondFirstPass Annotation • There was 1 potential operon found in both C58 and S4, with some interesting differences between them. In S4, the potential operon is hisB/H/A/F/E. In C58, there must have between an inversion and an insertion because the potential operon is sitting in the opposite direction from that seen in S4 and the operon consists of hisH/A/F/E. The hisB gene is just upstream of the operon, but now separated from it by the insertion of a novel gene in the opposite direction. • In addition to the gene movement mentioned above for one copy of hisC, there appears to have been a transfer of a piece from ChrI to ChrII in the biovar 3 lineage after its split from biovar 1. The transferred piece contains the hisG gene. L-Histidine

19. 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)

20. 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 Example #4 Testing Hypotheses Based on Sequence Annotation

21. 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

22. Functional Genomics Brainstorming & Experimental Design • Students hit the primary literature to learn about the enzymatic function encoded by their putative gene & how they might test it • Enzyme assays, growth curves, biochemical complementation, etc. are possible tests • Don’t reinvent the wheel, yet allow for creativity • Stress proper controls & repetition • Students provide a materials list & basic setup for their proposed experiment

23. Functional Genomics Constructing Gene Disruption Mutants • 67 genes disrupted since spring of 2002 by MolCell students • 40 genes encoding specific enzymes: multiple genes involved in sucrose metabolism 2 aconitases 4 malate dehydrogenases – only 2 with definable impact • 27 genes encoding two component systems (mostly response regulators): currently finishing up a massive screen of 23 mutants across 54 treatments (covering 12 different environmental variables)

24. Functional Genomics Example = Catalase • Catalyzes breakdown of hydrogen peroxide • Spectrophotometric enzyme assay possible, but students spent most of their time working out the procedure and the proper controls • Published work shows that catalase is essential for tumor induction by A. tumefaciens; our gene disruption mutant acted as expected wildtype catalase-

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

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

27. 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

28. Forward Genetic Screens 2006 Hiram Genomics Academy • Each session lasted 3-5 days • Students generated mutants, screened for phenotypes, recovered Tn insertion sites for sequencing, & learned some bioinformatics • 44 high school students + 11 Hiram students generated over 10K mutants, screened 8344 mutants for 10 different phenotypes, & identified 86 mutants worthy of further study • 44 students from 37 different high schools in OH, PA, MI, & IN spread over 3 summer sessions