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HiramGenomicsInitiative
Collaborations
Teaching
High schoolStudents
HiramStudents
Recruiting
Research
Chromohalobacter salexigens
(w/ Purdue Univ. & DOE-JGI)
Sphingomonas elodea(w/ Monsanto Co.)
Azotobacter vinelandii(NSF grant w/ 4 partners)
Agrobacteriumbv. 2 & 3 strains(NSF grant w/ 7
partners) 2 Xenorhabdus species
(USDA grant w/ 6 partners)
Hiram Genomics Initiative Agrobacterium Other Genome ProjectsGenome Project
Sphingomonas Chromohalobacter XenorhabdusAzotobacter
elodea salexigens bovienii & 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 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)
Gene Mutant Gap Sequence
Disruptions Screens 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
plant cell
bacterium
DNAhormones
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
Genome
Random Pieces Shotgun
GenomicLibraries
6-8XSequencingCoverage
Overlaps in Small Piecesto Form Contigs
Join LargePieces intoSequenced Genome
Genetic/Physical
Map
SubgenomicLibraries
1XSequencingCoverage
Annotation ofContig Ends
Gap Closure
Functional Genomics
Annotation
SubgenomicMega-fragments
Basics of a Genome Project
Genome
Random Pieces Shotgun
GenomicLibraries
6-8XSequencingCoverage
Overlaps in Small Piecesto Form Contigs
Join LargePieces intoSequenced Genome
Genetic/Physical
Map
SubgenomicLibraries
1XSequencingCoverage
Annotation ofContig Ends
Gap Closure
Functional Genomics
Annotation
SubgenomicMega-fragments
Example #1Generating Combined Genetic/Physical Map
Combined Genetic/Physical Maprich medium minimal medium
Transposonmutagenesis
Mutant screening (auxotrophs?) & characterization
Physical mapping (PFGE)
Recovery of Tn insertion site
CombinedGenetic &Physical
Maps
(J. Bact.181:5160-6)
Combined Genetic/Physical Map Connecting Sequence Contigs to Map (Tn5-RL27)
1 2 3 4
1: Digestion with SacII … dilute ligation2: Transform into pir+ E. coli3: Sequence off Tn ends … query contigs4: Contig can be placed on map
Genome
Random Pieces Shotgun
GenomicLibraries
6-8XSequencingCoverage
Overlaps in Small Piecesto Form Contigs
Join LargePieces intoSequenced Genome
Genetic/Physical
Map
SubgenomicLibraries
1XSequencingCoverage
Annotation ofContig Ends
Gap Closure
Functional Genomics
Annotation
SubgenomicMega-fragments
Example #2Bioinformatics-based Gap Closure
Bioinformatics-based Gap ClosureComparing the Ends of Contigs
Partner A Contig Partner 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
Bioinformatics-based Gap ClosureExamples from Sphingomonas elodea
Partner A Contig Putative Join Partner B ContigLeft 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 -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
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 ClosureUsing 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
Genome
Random Pieces Shotgun
GenomicLibraries
6-8XSequencingCoverage
Overlaps in Small Piecesto Form Contigs
Join LargePieces intoSequenced Genome
Genetic/Physical
Map
SubgenomicLibraries
1XSequencingCoverage
Annotation ofContig Ends
Gap Closure
Functional Genomics
Annotation
SubgenomicMega-fragments
Example #3Sequence 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
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.
BeyondFirstPass
AnnotationStudents as
Pathway Experts
L-Histidine
BeyondFirstPass
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.
L-Histidine
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.
Beyond 1st Pass AnnotationStudents as 2nd Pass Annotators
Figure 2. Isoelectric Points of Outer Membrane Proteins
3171
3071
3022
2860
2691
2485
2448
2171
2098
1580
1452
1443
1442
1394
1355
1139
1132
941
799
768
766
723
213
2
3
4
5
6
7
8
9
10
11
12
pI
C. salexigens
E. coli K12
P. aeruginosa PA01
Series4
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)
Genome
Random Pieces Shotgun
GenomicLibraries
6-8XSequencingCoverage
Overlaps in Small Piecesto Form Contigs
Join LargePieces intoSequenced Genome
Genetic/Physical
Map
SubgenomicLibraries
1XSequencingCoverage
Annotation ofContig Ends
Gap Closure
Functional Genomics
Annotation
SubgenomicMega-fragments
Example #4Testing Hypotheses Based on Sequence Annotation
• 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 portionplasmid pCR2.1 of gene of genecannot replicatein Agrobacterium Cbr
Functional GenomicsConstructing Gene Disruption Mutants
• 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 GenomicsBrainstorming & Experimental Design
• 67 genes disrupted since spring of 2002 by MolCell students
• 40 genes encoding specific enzymes:multiple genes involved in sucrose metabolism
2 aconitases4 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 GenomicsConstructing Gene Disruption Mutants
• 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 GenomicsExample = Catalase
wildtype
catalase-
Functional GenomicsExample = 2 Aconitases in Agrobacterium C58
wt acnA-
wildtype A. tumefaciensfrom LB plate (pH7)
A. tumefaciens acnA- mutantfrom LB plate (pH7)
• One group wanted to look at motility!?
• Motility is one process regulated post-transcriptionally by apo-AcnB in E. coli
Functional GenomicsForward Genetics Screens
Transposonmutagenesis
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 ScreensHigh 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
• 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 Screens2006 Hiram Genomics Academy
• 44 students from 37 different high schools in OH, PA, MI, & IN spread over 3 summer sessions