Transcript Document

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
(NSF grant w/ 4 partners)
Hiram
Students
High
school
Students
Recruiting
Hiram Genomics Initiative
Agrobacterium
Genome Project
Other Genome Projects
Sphingomonas
elodea
Functional Genomes of
Native Genomics of K84 (bv. 2)
Tumor Strain C58 & S4 (bv. 3)
Genetic/
Survey (biovar 1)
Physical Map
(high
schools)
(Genetics)
Chromohalobacter
Xenorhabdus
Azotobacter
salexigens bovienii & nematophila vinelandii
Genetic/
Physical Map
Genetic/
Physical Map
Gap
Closure
Genetic/
Physical Map
(Genetics)
(Genetics &
high schools)
(Independent
Research)
(Genetics &
Independent
Research)
Gap
Closure
(Independent
Research)
Sequence
Annotation
(MolCell, Genetics,
& Biochem)
Gene
Disruptions
(MolCell &
Independent
Research)
Mutant
Gap
Screens Closure
Sequence
Annotation
(MolCell & (Independent (Genetics &
Independent Research) Independent
Research)
Research)
Sequence
Annotation
(Independent
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
Example of Success:
Agrobacterium Genome Project
bacterium
hormones
DNA
plant cell
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
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
2
3
4
1: Digestion with SacII … dilute ligation
2: Transform into pir+ E. coli
3: Sequence off Tn ends … query contigs
4: 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)
Partner B Contig
Right end of C466
reading in
Right end of C448
reading in
Right end of C528 g-glutamyl-P reductase ORF (gap
reading out
is bases encoding AA#230-235)

Right end of C523
reading in
Left end of C502
reading out

Right end of C482
reading in
Ribonuclease R ORF(gap is bases
encoding AA#420-430)
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.
L-Histidine
• 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
L-Histidine
• 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
L-Histidine
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 Annotation
Students as 2nd Pass Annotators
12
C. salexigens
E. coli K12
P. aeruginosa PA01
11
Series4
10
9
8
pI
Chromohalobacter
salexigens annotation by
Biochem students to test
the hypothesis that proteins
in halophiles are more
acidic than their homologs
in nonhalophic relatives
7
6
- PSORT (cellular localization)
- BLAST (homologs in E. coli &
P. aeruginosa)
- MW/pI (pI determination)
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
• 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 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
wt
acnA-
5
Colony Swarming (cm diameter)
4.5
4
Hour 0
Hour 6.5
Hour 24
Hour 30.5
3.5
3
2.5
2
1.5
1
0.5
0
C58
(LB
0.3)
C58
(LB
0.6)
C58
(LB 1)
C58
(LB
1.5)
acnA(LB
0.3)
acnA(LB
0.6)
acnA(LB 1)
acnA(LB
1.5)
C58
(M9
0.3)
C58
(M9
0.6)
C58
(M9 1)
C58
(M9
1.5)
acnA(M9
0.3)
Strain
(M edium, % Agar)
wildtype A. tumefaciens
from LB plate (pH7)
A. tumefaciens acnA- mutant
from LB plate (pH7)
acnA(M9
0.6)
acnA(M9 1)
acnA(M9
1.5)
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