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Fuctional Genomics: Functional Complementation Project

Fuctional Genomics: Functional Complementation Project. Some Issues with Research in the Classroom. Difficult for instructors to identify/implement novel research projects each semester/year

kadeem-emerson
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Fuctional Genomics: Functional Complementation Project

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  1. Fuctional Genomics: Functional Complementation Project

  2. Some Issues with Research in the Classroom • Difficult for instructors to identify/implement novel research projects each semester/year • Planning/preparation time must be efficient (minimal) and easily accomplished by busy faculty or teaching assistants • Experiments must be well defined and capable of producing clear outcomes • Should address multiple scientific topics within a series of experiments

  3. How We are Working to Solve Them • We have designed the core of a curriculum, based on genetic complementation of defined E. coli mutants, that enables true experimentation in the classroom • The modular format allows testing completely different genes every semester but does so with repetitive sets of protocols and materials • Consistency makes it possible for busy faculty to perform actual research in the classroom without having to prepare de novo labs every semester

  4. Why use Complementation as a basis for curriculum modules? • In many cases, it provides a clear “life or death” result that can be easily interpreted. • Although intermediate phenotypes (e.g. slow growth) can be distinguished. • It lends itself to testing thousands of different types of genes. • Assays can be simple (survival or colorimetric), complex (GC analysis of metabolites) or anything in between

  5. Why use Complementation as a basis for curriculum modules? • The framework enables integration of many techniques and genetic principles • Bioinformatics (from simple BLAST through programming) • Auxotrophy vs. prototrophy; Epistasis • Basic molecular techniques such as PCR, cloning, selection, restriction endonuclease mapping, etc. • Gene induction and regulation • If desired, enzymatic analysis • Each experiment provides functional data that can be used to update annotation and construct publications

  6. Why use Complementation as a basis for curriculum modules? • Perhaps most importantly, it lends itself to highly repeatable experiments that are all variations on a theme • The vectors, techniques, and (most) instructional materials are consistent from semester to semester • The primary changes each year involve the particular pathway under investigation and the choice of genes to test

  7. We are Enabling the System Through Curriculum “Kits” • We aim to combine the engagement of original research with the straightforward techniques typical of “kits”, such as those for cloning GFP • Each kit contains: • A defined E. coli mutant and isogenic WT strain • A cloning vector • A positive-control plasmid containing the E. coli version of the gene • Complete protocols • Background information on the experiment • Support via a web site for downloading information, asking questions, uploading results, and connecting with other groups performing similar or identical sets of experiments.

  8. We developed a specific vector for the program Broad host-range (pBBR origin) Low copy number Amp resistant to avoid outgrowth after transformation sacB gene provides counter-selectable marker to remove background Arabinose-inducible expression of gene-of-interest Cloning site flanked by NotI sites Designed for ligase-independent cloning

  9. 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ no As no As x x x x x x x x x x x x ATG TAC TAAATT . . . . . . . . . . . GOI . . . . x x x x x x x x x x x x x x x RBS AGGAGGA x x x x x x x x x x x x x x x 5’ AGGAGGA NotI sacB NotI 3’ araC Promoter TCCTCCT x x x x x x x x x x x x TCCTCCT vector vector no Ts ~12 nts no Ts ~ 15 nts Plasmid Kt-1 5246bp ampr xxxxxxx = region of homology; provides sufficient overlap for hybridization of insert and vector for LIC RBS = ribosome binding site ccdB= cytotoxic gene; when present in E. coli, will kill the bacterium nts = nucleotides ampr= ampicillin resistance gene araC promoter = arabinose promoter; weak; necessary for gene expression LIC Base Vector and Overview araC

  10. Ligation Independent Cloning no As 5’CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC 3’ GCTGTTCTCGCCGGCGTACCTTTTCTTTTAGCCAAAATAACCG CTCAGCAAATCCTGATGAGGCCGCTTGGTGTT 3’ GAGTCGTTTACCACTACT CCGGCGAACCACAA5’ proC insert no As . . . . . . . . . . . . . . . . . . . . weak RBS no Ts GGACAATTAACAGTTAACAAATAA GCGGCCGCTTGGTGTTTCTAGAATCATG -3’ CCTGTTAATTGTCAATTGTTTATT CGCCGGCGAACCACAAAGATCTTAGTAC-5’ 5’GAATTCGACAAGAGCGGCCGCATGAACATCAAAAAGTTTGC 3’CTTAAGCTGTTCTCGCCGGCG TACTTGTAGTTTTTCAAACG sacB vector vector ERI NotI NotI no Ts 5’-GGCCGCTTGGTGTTTCTAGA-3’ 3’-CGAACCACAAAGATCT-5’ 5’-GAATTCGACAAGAGC-3’ 3’-CTTAAGCTGTTCTCGCCGG-5’ digest vector with NotI vector vector NotI XbaI ERI NotI dTTP Treat vector and insert with T4 DNA Polymerase XbaI 5’-GGCCGCTTGGTGTTTCTAGA3’ 3’-TCT5’ vector dATP 5’ CGACAAGAGCGGCCGC ATGGAAAAGAAAATCGGTTTTATTGGC 3’ ACCTTTTCTTTTAGCCAAAATAACCG CTCAGCAAATCCTGATGA 3’ GAGTCGTTTACCACTACT CCGGCGGAACCACAA-5’ proC dATP 5’GAATT 3’CTTAAGCTGTTCTCGCCGG vector ERI NotI dTTP T4 DNA Polymerase 3’ 5’ exonuclease digests DNA until the first specified nucleotide (A or T) is reached. T4 DNA Polymerase idles at the A or T since the enzyme defaults to the polymerizing activity when dATP or dTTP is supplemented into the respective reaction.

  11. Our first kits were built around Amino Acid Biosynthetic Pathways Proline Genes proA proB proC Arginine Genes argA argB argC argD argE argF argG argH argI carA carB Aspargine/ Isoleucine Genes asnA asnB ilvA ilvC ilvD ilvE ilvBN ilvGM ilvIH Glutamine/ Ammonia AssimilationGenes glnA glnB glnD glnE glnG glnL ropN

  12. Agrobacterium tumefaciens C58: Background • Member of Rhizobiaceae • Causes crown gall disease in dicots. • Infection mediated by transforming plant cells with tumor inducing plasmid (Ti). • Uses virulence proteins to recognize and attach to plant cells. • Fully sequenced genome. http://www.chemistry.emory.edu/faculty/lynn/research/agro/viravirg.jpg http://m1.ikiwq.com/img/xl/5JH2txljqSDGA4Vi1QrNya.jpg Wood, et al. 2001.

  13. Proline Biosynthesis: The common pathway • proA (Atu 2779) • Glutamate-γ-semialdehyde dehydrogenase • Circular chromosome • proB (Atu 2780) • Glutamyl kinase • Circular chromosome • proC1 (Atu 2209) • Pyrroline-5-carboxylate reductase • Circular chromosome • proC2 (Atu 2985) • Pyrroline-5-carboxylate reductase • Linear chromosome Deutch, et al. 1984

  14. Rhizobiaceae phylogeny A. tumefaciens C58 201262 codons Circular chromosome proC1 proA proB Linear chromosome proC2 B. japonicum 2575793 codons proB proA proC R. etli Circular chromosome 384927 codons Rhizobium etli proC1 proB proA Plasmid proC2 Hayzer, et al. 1980. González, et al. 2006. 699677 codons M. loti proA proB proC

  15. Results: Complementation analysis

  16. The Arginine Biosynthetic Pathway From: Xu, Ying et. al. Microbiology and Molecular Biology Reviews, Mar 2007 71(1): 36-47.

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