Transcript Document
Basics of a Genome Project The Sequence is Not the End of the Road Overlaps in Small Pieces to Form Contigs Genome 8-20X Sequencing Coverage Gap Closure Random Pieces Shotgun Genomic Libraries Sequencing without Cloning Join Large Pieces into Sequenced Genome Annotation Functional Genomics Functional Genomics Connecting Genes to Functions Overlaps in Small Pieces to Form Contigs Genome 8-20X Sequencing Coverage Gap Closure Random Pieces Shotgun Genomic Libraries Sequencing without Cloning Join Large Pieces into Sequenced Genome Annotation Functional Genomics Why Functional Genomics? Students Connecting Genes to Functions GENE FUNCTION Forward Genetics Functional Complementation Protein Overexpression Reverse Genetics Forward Genetics Transposon Mutagenesis in Genetics Course Transposon mutagenesis Mutant screening & characterization Recovery of Tn insertion site Sequence off of Tn end to identify mutated gene Forward Genetics Students Filling in Holes in Annotation These 2 functions are done by one bifunctional enzyme in E. coli (HisB), but not in Agrobacterium Imidazoleglycerolphosphate dehydratase domain of E. coli HisB picks up ortholog in Agrobacterium Forward Genetics Students Filling in Holes in Annotation Have a Tn hit in gene Atu3885: - annotated as myo-inositol monophosphatase family member - mutation leads to auxotroph - now know it cannot make histidine - TIGR Fam hit to alternative histidinol-P phosphatase … now proven through experimentation These 2 functions are done by one bifunctional enzyme in E. coli (HisB), but not in Agrobacterium Imidazoleglycerolphosphate dehydratase domain of E. coli HisB picks up ortholog in Agrobacterium Getting Transposon into Host Cells Tn chromosome Getting Transposon into Host Cells Transformation Tn Tn Tn Tn Tn chromosome Getting Transposon into Host Cells Transduction Tn chromosome Tn Getting DNA into Cells Conjugation Transposon Mutagenesis Use Transposon as a Tool Kanr oriR6K Tn5-RL27 hyperactive Tn5 transposase Transposon Mutagenesis Generating Transposon Hop Mutants Kanr Kanr ser- Transposon Mutagenesis Screens for Mutants rich minimal - 3-phosphoglycerate Screened ~11,000 Kanr colonies 103 auxotrophic mutants 3-phosphohydroxypyruvate - 3-phosphoserine - serine + glycine + Transposon Mutagenesis Be Creative with Screens (or Selections) rich minimal ? Minimal w/ different C or N sources – utilization mutants Rich or minimal at various pH – acid or base tolerance Rich with different [salt] – osmotic regulation mutants Rich with lower [agar] – motility mutants Rich – salt + dyes – biofilm mutants Transposon Mutagenesis Cloning Out Tn Insertions 1 2 3 4 1: Digestion with SacII … dilute ligation 2: Transform into pir+ E. coli 3: Sequence off Tn ends … query genome 4: Figure out how to connect gene to function Agrobacterium tumefaciens bacterium hormones DNA plant cell Agrobacterium tumefaciens bacterium DNA plant cell food Agrobacterium tumefaciens bacterium novel DNA plant cell Transgenic Plants