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Microbiology and Molecular Biology for Engineers

Microbiology and Molecular Biology for Engineers. IGEM, 20 June 2006. There are three types of cell. A: Archaea B: Bacteria (Gram positive, Gram negative) E: Eukarya (Animals, plants, yeasts, others). Eschericha coli. Kingdom/Division Proteobacteria Class/Subdivision Gammaproteobacteria

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Microbiology and Molecular Biology for Engineers

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  1. Microbiology and Molecular Biology for Engineers IGEM, 20 June 2006

  2. There are three types of cell • A: Archaea • B: Bacteria (Gram positive, Gram negative) • E: Eukarya (Animals, plants, yeasts, others)

  3. Eschericha coli • Kingdom/Division Proteobacteria • Class/Subdivision Gammaproteobacteria • Family Enterobacteriaceae sensors communications hull propulsion control systems power plant

  4. Cell envelope • Typical of Gram negative bacteria • Outer membrane: repels hydrophobic molecules. • Peptidoglycan sacculus: resists osmotic pressure. • Cell membrane: main permeability barrier.

  5. Energy generation • Energy generated by oxidation or disproportionation of organic molecules. • Stored as non-equilibrium ATP/ADP ratio and trans-membrane proton gradient. Photon capture Respiration Fermentation Motility Transport processes DNA, RNA and protein synthesis Trans-membrane proton gradient ATP/ADP ratio F1F0 ATPase

  6. Motility • Swimming motility: rotating flagella powered by proton influx. • Helical filaments: 20 nm diameter, 5 to 20 mm long. filament hook (Gram negative bacteria only) OM L-ring P-ring PG EM of flagellar base structure CM MS-ring motor protein switch protein

  7. Two-component sensor systems • Sense external stimuli. • External stimulus causes modification of internal protein. ligand PERIPLASM Cell membrane Sensor Kinase CYTOPLASM Response Regulator P

  8. Chemotaxis • Swimming towards an attractant or away from a repellant is accomplished by a biased random walk – variable length runs interspersed with random changes of direction (tumbles).

  9. Mechanism of chemotaxis • Attractant / repellent chemicals are detected by chemotaxis receptors (MCPs). • Phosphorylation state of CheY alters frequency of tumbles. • Methylation of MCPs decreases sensitivity. methylase CheY phosphatase Glu-Me CheY P promotes tumbling ligand MCP CheA CheB CheW phosphatase demethylase: P CheB increases MCP sensitivity

  10. Intercellular communication • Cells sense population density by ‘quorum sensing’. • Detect critical density of an autoinducer, usually a homoserine lactone (LuxI/LuxR-type system) Picture of squid O N O OHHL O O LuxR luxI luxC luxD luxA luxB luxE luxG lux promoter

  11. Life cycle 1: shaken broth cultures • Exponential growth phase followed by stationary phase with different genes expressed. Biomass/ Optical density exponential phase stationary phase decline phase lag phase

  12. Life Cycle 2: in nature • Cells prefer to grow attached at solid-liquid interfaces (biofilm). detachment swimming cells swimming motility, chemotaxis surface-associated motility quorum sensing attached cells microcolonies mature biofilm

  13. Stochastic vs. mean field models • Simplistic models often treat biomass as a single compartment. • More realistically, billions of individual cells which may be in quite different states. • Therefore, oscillators etc. must include a cell synchronization mechanism unless individual cells are to be monitored (eg by FACS or fluorescence microscopy).

  14. Hosts other than E. coli • Salmonella: related to E. coli but better secretion of proteins into the medium. Problem: pathogen. • Bacillus: good secretion of proteins, forms highly stable resting state (endospores). • Budding yeast (Saccharomyces cerevisiae): eukaryotic cells. • Plant, insect and mammalian cells. endospores yeast

  15. Coming up… • What the stuff inside the cell is made of, and how it works. • How to modify it.

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