Closure (Independent Sequence Sequence Research) Annotation Annotation (MolCell, Genetics, (Independent & Biochem) Research) Gene Mutant Gap Sequence 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 food plant cell 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 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 1: 2: 3: 4: 2 3 4 Digestion with SacII dilute ligation Transform into pir+ E. coli Sequence off Tn ends query contigs 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) Right end of C528 -glutamyl-P reductase ORF (gap reading out is bases encoding AA#230-235) Left end of C502 reading out Ribonuclease R ORF(gap is bases encoding AA#420-430)
Partner B Contig Right end of C466 reading in Right end of C448 reading in Right end of C523 reading in Right end of C482 reading in 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. LHistidine 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 LHistidine 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 hisB/H/ LA/F/E. In C58, there must have between an inversion and an insertion Histidine 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 pI Chromohalobacter salexigens annotation by Biochem students to
test the hypothesis that proteins in halophiles are more acidic than their - PSORT (cellular homologs in localization) nonhalophic - BLAST (homologs in E. coli relatives & P. aeruginosa) - MW/pI (pI determination) C. salexigens E. coli K12 P. aeruginosa PA01 11 Series4 10 9 8
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 Dont 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 wildtype A. tumefaciens from LB plate (pH7) wt A. tumefaciens acnA- mutant from LB plate (pH7) acnA- 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
2018-19 SY Funding. Third year for the grant. The distribution of this award is for the implementation of the goals of the grant which are carried out by the activities identified in the school's approved application and signed assurances. No...
Dr. John Long Middle School Home of the Longhorns! ... each Courtyard time after lunch No assigned seats and opportunities to eat outside Tools for Middle School Success eSembler CANVAS Planner/Photo of board Organization - Clean out backpack once a...
These are some of the skills we have already covered at least once in class: You will also have covered many of these skills in GCSE Maths and Geography Annotating diagrams, sketches, photos, graphs etc. Overlays . Literacy and numeracy...
Acknowledgments Therapy of human rabies: lessons from experimental studies in a mouse model Alan C. Jackson, MD Courtney A. Scott, BSc James Owen, BSc Simon C. Weli, PhD John P. Rossiter, MB, PhD Departments of Medicine (Neurology) Microbiology and Immunology...
When I grow up, I want to be a little boy. Joseph Heller Nowadays people know the price of everything and the value of nothing. Oscar Wilde There isn't any answer. ... They also like the fun of seeing how...
The Present Scope of the Common Law. Today, the common law (judge-made law) still governs certain areas; contracts, torts, and property are largely uncodified and remain subject to the common law. Even in those fields of law, however, federal and...
Social housing is affordable housing for people on low incomes, usually managed by local authorities or housing associations. When there is a not a permanent property available, those eligible receive temporary accommodation (TA) - which can last for years. There...
AO- Kursio Nerija National Park Administration. WTPB.02.01.00-62-014/10. Management of Rosa rugosa along the South Baltic coast (RosaRugosa) National Forest and Nature Agency, dept. Storstroem. State Agency for Environment and Nature Rostock. Kursiu Nerija National Park Administration. University of Rostock,
Ready to download the document? Go ahead and hit continue!