Marrying the Classic and Contemporary

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Marrying the Classic and Contemporary in
Microbial Genetics
Joel Griffitts Brigham Young University
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Brigham Young University Undergraduate
Enrollment 29,000 Life Science 2,600 Health and
Human Performance 2,500 Nursing 900
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The classical approach to studying the genetics
of symbiosis
Sinorhizobium meliloti (wt) on alfalfa
Mutant R3H4 on alfalfa
(Fix)
(Fix-)
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A new kind of mutant
Gain-of-compatibility mutant
Naturally incompatible strain inoculated on M.
praecox
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Daniel Lindsay
Divyesh Choudhri
Joanna Ryther
Matt Crook
Ryan VanYperen
Elise Scoggin
Clarice Harrison
Lejdja Beshku
Rebecca Elliott
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Dis?cov?er (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
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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
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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)
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rIIA
rIIB
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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
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Our Tool Kit
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/Fluores
cent protein reporters Microarrays Massively
parallel sequencing
Our Heritage
The Genetic Code DNA replication Mutation Recombin
ation Transposition Transformation Conjugation Tra
nsduction Gene regulation Plasmids
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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
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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

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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

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Structure/Function of the Cell Life without walls
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Making a wall-less strain
Pxyl-murE
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Wall-less
Wild type
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Defense Against Parasites CRISPR
CRISPRClustered Regularly Interspersed
Palindromic Repeat
(Streptococcus thermophilus)
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(CRISPR in Strep thermophilus)
Select for resistance to phage F858 or F2972
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CRISPR cassettes can block all sorts of parasitic
DNAs
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Intercellular Communication The Extracellular
Death Factor
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Toxins/Antitoxins in Plasmid Addiction
Toxin
Antitoxin
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The Riddle of Chromosomal Toxin-antitoxin Systems
mazEF
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The Extracellular Death Factor (EDF)
Modulates Programmed Cell Death Via the mazEF TA
System
N
N
W
N
N
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Horizontal Gene Transfer Conjugation Made
Explicit
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Visualizing Gene Transfer The Strategy
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tetR/Ptet-RFP
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seqA-yfp
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SeqA-YFP
RFP (leaky)
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Synthetic Genomics
Phase 1 Synthesizing a whole chromosome
Phase 2 Re-booting a cell
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Phase 1 Synthesizing a whole chromosome
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Phase 2 Re-booting a cell
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Mycoplasma mycoides
Mycoplasma capricolum
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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
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