What a Great Time to Teach & Do Research with Undergrads!! So much data! *** So much for me & my students to do!!!*** So many questions! So many great tools! Too few researchers! As of 1/3/08: DomainPublished Ongoing Archaea5068 Bacteria Eucarya77891 (+ 114 metagenomes + GEBA plan)

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) Ammonifex degensii (JGI pilot program w/ 10 partners)

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)

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

Basics of a Genome Project

Opportunity Sequence Annotation

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

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)

Opportunity Testing Bioinformatics Predictions

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

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) Functional Genomics Constructing Gene Disruption Mutants

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

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

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

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 Forward Genetic Screens Hiram Genomics Academy 79 students from high schools in OH, PA, MI, & IN spread over 5 summer sessions in 2006 & 2007