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