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