Teaching Research Collaborations Agrobacterium bv. 2 & 3

Teaching Research Collaborations Agrobacterium bv. 2 & 3

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

school Students Recruiting Hiram Genomics Initiative Agrobacterium Genome Project Other Genome Projects Sphingomonas Chromohalobacter Xenorhabdus Azotobacter elodea nematophila salexigens bovienii & vinelandii Functional Genomes of Native Genomics of K84 (bv. 2) Tumor Strain C58 & S4 (bv. 3)

Genetic/ Gap Genetic/ Survey (biovar 1) Physical Map Closure Physical Map (high (Independent (Genetics & schools) schools) Research) Independent (Genetics) Genetic/ Physical Map Genetic/ Physical Map (Genetics) (Genetics & high Gap Research)

Closure (Independent Sequence Sequence Research) Annotation Annotation (MolCell, Genetics, (Independent & Biochem) Research) Gene Mutant Gap Sequence 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 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 Basics of a Genome Project Subgenomic Mega-fragments Genome Subgenomic Libraries 6-8X Sequencing Coverage Overlaps in Small Pieces to Form Contigs Gap Closure

Random Pieces Shotgun Genomic Libraries Join Large Pieces into Sequenced Genome Annotation Functional Genomics 1X Sequencing Coverage Genetic/ Physical Map Annotation of Contig Ends Example #1 Generating Combined Genetic/Physical Map Subgenomic

Mega-fragments Genome Subgenomic Libraries 6-8X Sequencing Coverage Overlaps in Small Pieces to Form Contigs Gap Closure Random Pieces Shotgun Genomic Libraries Join Large Pieces into Sequenced Genome Annotation Functional Genomics

1X Sequencing Coverage Genetic/ Physical Map Annotation of Contig Ends Combined Genetic/Physical Map rich medium Transposon mutagenesis Recovery of Tn insertion site minimal medium Mutant screening (auxotrophs?) & characterization Physical mapping

(PFGE) Combined Genetic & Physical Maps (J. Bact. 181:5160-6) Combined Genetic/Physical Map Connecting Sequence Contigs to Map (Tn5-RL27) 1 1: 2: 3: 4: 2 3 4 Digestion with SacII dilute ligation Transform into pir+ E. coli Sequence off Tn ends query contigs Contig can be placed on map

Example #2 Bioinformatics-based Gap Closure Subgenomic Mega-fragments Genome Subgenomic Libraries 6-8X Sequencing Coverage Overlaps in Small Pieces to Form Contigs Gap Closure Random Pieces Shotgun Genomic Libraries Join Large Pieces into

Sequenced Genome Annotation Functional Genomics 1X Sequencing Coverage Genetic/ Physical Map Annotation of Contig Ends Bioinformatics-based Gap Closure Comparing the Ends of Contigs Gene X? Partner A Contig BLAST analysis of the right end of contig A reveals the first part of gene X Partner B Contig 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 Bioinformatics-based Gap Closure Examples from Sphingomonas elodea Partner A Contig Putative Join Left end of C452 Glucokinase ORF (gap is a few reading out bases near codon for AA#71) Right end of C491 reading out cobW ORF (gap is bases encoding AA#120-500) Right end of C528 -glutamyl-P reductase ORF (gap reading out is bases encoding AA#230-235) Left end of C502 reading out Ribonuclease R ORF(gap is bases encoding AA#420-430)

Partner B Contig Right end of C466 reading in Right end of C448 reading in Right end of C523 reading in Right end of C482 reading in Bioinformatics-based Gap Closure Using One Genome to Close Another Query Contig from A. tumefaciens C58 500 500 bp 500 Subject Contig 1 from A. vitis S4

500 500 bp 500 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 Example #3 Sequence Annotation Subgenomic Mega-fragments Genome Subgenomic Libraries 6-8X Sequencing Coverage

Overlaps in Small Pieces to Form Contigs Gap Closure Random Pieces Shotgun Genomic Libraries Join Large Pieces into Sequenced Genome Annotation Functional Genomics 1X Sequencing Coverage Genetic/ Physical Map Annotation of Contig Ends

Annotation Pipeline 0 kb 10 kb 20 kb 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) Beyond First Pass Annotation Students as Pathway Experts

Genetics students assigned a pathway to compare 2 strains of Agrobacterium in terms of gene content, gene order, etc. LHistidine 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 LHistidine 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. 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/ LA/F/E. In C58, there must have between an inversion and an insertion Histidine 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. Beyond 1st Pass Annotation Students as 2nd Pass Annotators 12 pI Chromohalobacter salexigens annotation by Biochem students to

test the hypothesis that proteins in halophiles are more acidic than their - PSORT (cellular homologs in localization) nonhalophic - BLAST (homologs in E. coli relatives & P. aeruginosa) - MW/pI (pI determination) C. salexigens E. coli K12 P. aeruginosa PA01 11 Series4 10 9 8

7 6 5 4 3 3171 3071 3022 2860 2691 2485 2448 2171 2098

1580 1452 1443 1442 1394 1355 1139 1132 941 799 768 766 723 213 2

Figure 2. Isoelectric Points of Outer Membrane Proteins Example #4 Testing Hypotheses Based on Sequence Annotation Subgenomic Mega-fragments Genome Subgenomic Libraries 6-8X Sequencing Coverage Overlaps in Small Pieces to Form Contigs Gap Closure Random Pieces Shotgun Genomic Libraries

Join Large Pieces into Sequenced Genome Annotation Functional Genomics 1X Sequencing Coverage Genetic/ Physical Map Annotation of Contig Ends 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 gene of interest in A. tumefaciens genome plasmid pCR2.1 cannot replicate in Agrobacterium

portion of gene Cbr plasmid portion of gene Cbr Clone PCR product & confirm by restriction mapping Introduce cloned PCR product into wildtype and select for single crossover gene disruption 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 Dont reinvent the wheel, yet allow for creativity Stress proper controls & repetition Students provide a materials list & basic setup for their proposed experiment 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) 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- Functional Genomics

Example = 2 Aconitases in Agrobacterium C58 One group wanted to look at motility!? Motility is one process regulated posttranscriptionally by apo-AcnB in E. coli wildtype A. tumefaciens from LB plate (pH7) wt A. tumefaciens acnA- mutant from LB plate (pH7) acnA- Functional Genomics Forward Genetics Screens Transposon mutagenesis Sequence off of Tn end to identify mutated gene Mutant screening & characterization Recovery of Tn insertion site Forward Genetic Screens High School Students Can Do It

Real world = multiple classes since 2002 from 5 area high schools 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 2006 Hiram Genomics Academy 44 students from 37 different high schools in OH, PA, MI, & IN spread over 3 summer sessions 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

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