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

2. Hiram Genomics Initiative Agrobacterium Other Genome Projects Genome Project SphingomonasChromohalobacter XenorhabdusAzotobacter elodeasalexigens bovienii & nematophila vinelandii Functional Genomes of NativeGenomics of K84 (bv. 2) Tumor Strain C58 & S4 (bv. 3)Genetic/Genetic/Genetic/GapGenetic/ Survey (biovar 1) Physical MapPhysical MapPhysical MapClosure Physical Map (high (Genetics) (Genetics) (Genetics & (Independent(Genetics & schools) high schools)Research)Independent Gap Research) Closure (Independent Sequence Sequence Research) Annotation Annotation (MolCell, Genetics,(Independent & Biochem)Research) GeneMutant Gap Sequence DisruptionsScreens Closure Annotation (MolCell & (MolCell &(Independent (Genetics & Independent Independent Research) Independent Research) Research) Research)

3. Bridging the Teaching-Research Gap Within Undergraduate Courses 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

4. plant cell bacterium DNA hormones Example of Success: Agrobacterium Genome Project food 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. Basics of a Genome Project

6. Example #1 Generating Combined Genetic/Physical Map

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

8. Combined Genetic & Physical Maps (J. Bact. 181:5160-6)

9. Combined Genetic/Physical Map Connecting Sequence Contigs to Map (Tn5-RL27) 123 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. Example #2 Bioinformatics-based Gap Closure

11. Bioinformatics-based Gap Closure Comparing the Ends of Contigs Partner A ContigPartner B Contig Gene X? 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 Design PCR primers (one reading off each end) & use them to amplify the missing gap sequence

12. Bioinformatics-based Gap Closure Examples from Sphingomonas elodea Partner A ContigPutative Join Partner B Contig Left end of C452Glucokinase 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  -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. Subject Contig 1 from A. vitis S4 Query Contig from A. tumefaciens C58 500 500 bp 500 Subject Contig 2 from A. vitis S4 500 500 bp 500 Bioinformatics-based Gap Closure Using One Genome to Close Another 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. Example #3 Sequence Annotation

15. 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) 0 kb 10 kb 20 kb

16. L-Histidine Genetics students assigned a pathway to compare 2 strains of Agrobacterium in terms of gene content, gene order, etc. 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. Beyond First Pass Annotation Students as Pathway Experts

17. L-Histidine Beyond First Pass Annotation 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. L-Histidine Beyond First Pass 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.

19. Beyond 1 st Pass Annotation Students as 2 nd 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. Example #4 Testing Hypotheses Based on Sequence Annotation

21. 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 Cb r portion plasmid portion plasmid pCR2.1 of gene of gene cannot replicate in Agrobacterium Cb r Functional Genomics Constructing Gene Disruption Mutants

22. 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 Functional Genomics Brainstorming & Experimental Design

23. 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) Functional Genomics Constructing Gene Disruption Mutants

24. 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 Functional Genomics Example = Catalase wildtype catalase -

25. Functional Genomics Example = 2 Aconitases in Agrobacterium C58 wt acnA - wildtype A. tumefaciens from LB plate (pH7) A. tumefaciens acnA- mutant from LB plate (pH7) 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 Sequence off of Tn end to identify mutated gene Recovery of Tn insertion site

27. 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 Forward Genetic Screens High School Students Can Do It Real world = multiple classes since 2002 from 5 area high schools

28. 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 Forward Genetic Screens 2006 Hiram Genomics Academy 44 students from 37 different high schools in OH, PA, MI, & IN spread over 3 summer sessions