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Marrying the Classic and Contemporary in Microbial Genetics Joel Griffitts Brigham Young University Brigham Young University Undergraduate Enrollment: 29,000 Life Science: 2,600 Health and Human Performance: 2,500 Nursing: 900 NH4+ N N The classical approach to studying the genetics of symbiosis Sinorhizobium meliloti (wt) on alfalfa (Fix+) Mutant R3H4 on alfalfa (Fix-) A new kind of mutant Gain-of-compatibility mutant Naturally incompatible strain inoculated on M. praecox Why are we here? Divyesh Choudhri ... Daniel Lindsay Ryan VanYperen Joanna Matt Ryther Crook Lejdja Beshku Elise Scoggin Rebecca Elliott Clarice Harrison Discover (v) To obtain knowledge or awareness of something not known before; to make visible; bring to view Attributes of a successful discoverer: 1. Appreciation for seminal discoveries of the past 2. Proficiency with the tools of discovery 3. Familiarity with the current frontiers of discovery 4. Passionate, observant, contemplative, imaginative Attributes of a successful discoverer: 1. Appreciation for seminal discoveries of the past 2. Proficiency with the tools of discovery 3. Familiarity with the current frontiers of discovery 4. Passionate, observant, contemplative, imaginative Leaders in the Revolution in Molecular Biology (1943-1963) Avery, Oswald (1877-1955) Benzer, Seymour (1921-2007) Brenner, Sidney (b. 1927) Chargaff, Erwin (1905-2002) Chase, Martha (1927-2003) Crick, Francis (1916-2004) Delbruck, Max (1906-1981) Franklin, Rosalind (1920-1958) Hershey, Alfred (1908-1997) Jacob, Francios (b. 1920) Kornberg, Arthur (1918-2007) Lederberg, Joshua (1925-2008) Luria, Salvador (1912-1991) Meselson, Matthew (b. 1930) Monod, Jacques (1910-1976) Nirenberg, Marshall (b. 1927) Pauling, Linus (1901-1994) Perutz, Max (1914-2002) Sanger, Frederick (b. 1918) Stahl, Franklin (b. 1929) Tatum, Edward (1909-1975) Watson, James (b. 1928) Yanofsky, Charles (b. 1925) rIIA rIIB Attributes of a successful discoverer: 1. Appreciation for seminal discoveries of the past 2. Proficiency with the tools of discovery 3. Familiarity with the current frontiers of discovery 4. Passionate, observant, contemplative, imaginative Our Tool Kit Our Heritage The Genetic Code DNA replication Mutation Recombination Transposition Transformation Conjugation Transduction Gene regulation Plasmids Chemical mutagens PCR cloning PCR mutagenesis Mobilizable plasmids Suicide/Ts plasmids Internal fragment disruption Marker exchange by +/- selection Lambda-Red recombination Mini-transposons Transductional mapping Inducible expression plasmids lacZ/Fluorescent protein reporters Microarrays Massively parallel sequencing Attributes of a successful discoverer: 1. Appreciation for seminal discoveries of the past 2. Proficiency with the tools of discovery 3. Familiarity with the current frontiers of discovery 4. Passionate, observant, contemplative, imaginative Bacterial Genetics at the Cutting Edge • Structure/Function of the Cell • Shape • Envelope maintenance • RNA-mediated Regulation • Riboswitches • Small RNAs • Defense Against Insults • CRISPR • Persisters • Programmed cell death/TA systems • Intercellular communication • Cooperation • Territoriality • w/ host cells • Horizontal Gene Transfer • Visualization • Regulation • Functional Genomics • Single-gene knock-out collections • Genetic interaction arrays • Synthetic Genomics • In vitro genome synthesis • Genome transplantation • Metabolic Engineering • More esoteric, but no less interesting: • Circadian (clock) biology • Appendage-less swimming • Magnet biosynthesis • Bdellovibrio predation • etc. Bacterial Genetics at the Cutting Edge • Structure/Function of the Cell • Shape • Envelope maintenance • RNA-mediated Regulation • Riboswitches • Small RNAs • Defense Against Insults • CRISPR • Persisters • Programmed cell death/TA systems • Intercellular communication • Cooperation • Territoriality •w/ host cells • Horizontal Gene Transfer • Visualization • Regulation • Functional Genomics • Single-gene knock-out collections • Genetic interaction arrays • Synthetic Genomics • In vitro genome synthesis • Genome transplantation • Metabolic Engineering • More esoteric, but no less interesting: • Circadian (clock) biology • Appendage-less swimming • Magnet biosynthesis •Bdellovibrio predation • etc. Structure/Function of the Cell: Life without walls Making a wall-less strain: Pxyl-murE + xylose no xylose Some wall-less progeny (L-forms) able to survive and proliferate Wall-less Wild type Defense Against Parasites: CRISPR CRISPR=Clustered Regularly Interspersed Palindromic Repeat (Streptococcus thermophilus) (CRISPR in Strep thermophilus) Select for resistance to phage Φ858 or Φ2972 CRISPR cassettes can block all sorts of parasitic DNAs Streptococcus thermophilus Escherichia coli Staphylococcus epidermidis Intercellular Communication: The Extracellular Death Factor Toxins/Antitoxins in Plasmid Addiction Toxin Antitoxin unstable Toxin stable Plasmid loss Toxin The Riddle of Chromosomal Toxin-antitoxin Systems I II mazEF Low dose of rif. Dense culture 90% dead mazEF Low dose of rif. Dilute culture 0% dead Low dose of rif. Dense culture 10% dead ΔmazEF III mazEF IV Low dose of rif. Dilute culture + Dense culture supernatant 95% dead Contains Death Factor The Extracellular Death Factor (EDF) Modulates Programmed Cell Death Via the mazEF TA System N N W N N Horizontal Gene Transfer: Conjugation Made Explicit (1946) Visualizing Gene Transfer: The Strategy tetR/P. tet-RFP . . . . .Hfr. . . . . .. . . + . dam . . . . . . .. . . . . .. . . . . . .. .. . .. . seqA-yfp dam- =SeqA-YFP =RFP (leaky) . . .. . . . . . . .. . . .. . . . .. . . . . .. . . .. . . .. .. . .. . . . . . . . . ... Synthetic Genomics Phase 1: Synthesizing a whole chromosome Phase 2: Re-booting a cell Phase 1: Synthesizing a whole chromosome Phase 2: Re-booting a cell Mycoplasma mycoides Mycoplasma capricolum Mycoplasma capricolum/mycoides Mycoplasma mycoides 1. Appreciation for seminal discoveries of the past 2. Proficiency with the tools of discovery 3. Familiarity with the current frontiers of discovery 4. Passionate, observant, contemplative, imaginative