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:
Domain Published Ongoing
Archaea 50 68
Bacteria 577 1628
Eucarya 77 891
(+ 114 metagenomes + GEBA plan)
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)Ammonifex degensii
(JGI pilot program w/ 10 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
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
OpportunitySequence 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 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
OpportunityTesting 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
Cbr
portion plasmid portionplasmid pCR2.1 of gene of genecannot replicatein Agrobacterium Cbr
Functional GenomicsConstructing 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 aconitases4 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 GenomicsConstructing Gene Disruption Mutants
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
• high school students + 11 Hiram students generated & screened 10,500 mutants for 10 different phenotypes, & identified 110 mutants worthy of further study
Forward Genetic ScreensHiram Genomics Academy
• 79 students from high schools in OH, PA, MI, & IN spread over 5 summer sessions in 2006 & 2007
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