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Cell Physiology

Cell Physiology. ChE 545. Five Kingdom Classification. Eukaryotic Cells. Procaryotic Cells. Eukaryote vs Prokaryotes - Physiology. Eukaryotic Cells Nuclear membrane – true nucleus Have several chromosomes Have organelles Have 80 S ribosomes Cell division is by mitosis

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Cell Physiology

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  1. Cell Physiology ChE 545

  2. Five Kingdom Classification

  3. Eukaryotic Cells Procaryotic Cells Eukaryote vs Prokaryotes - Physiology

  4. Eukaryotic Cells Nuclear membrane – true nucleus Have several chromosomes Have organelles Have 80 S ribosomes Cell division is by mitosis More complex structures Procaryotic Cells Nuclear region –no membrane Has one chromosome Has no organelles Have 70 S (smaller) ribosomes Cell division by binary fission Simpler structures Eukaryote vs Prokaryotes - Physiology

  5. Eukaryotic Cells Grow relatively slowly (double once per hour to day) Need more complex media Larger in diameter (smallest are 5 um or so) More difficult genetic manipulation few plasmids Make more different and more complex products Low producers (10’s mg/L) Procaryotic Cells Grow quickly – can double in less than 20 min Can grow on simple media Smaller cells (smallest are 0.3 um) Often easy genetic manipulation –many plasmids. Simple products High producers (esp proteins (g/L)) Eukaryote vs Prokaryotes - Biotech

  6. Prokaryotic Cells • Escherichia coli

  7. Prokaryotic Cells • Bacillus sps. • (subtillus, thuringiensis, anthracia)

  8. Ascomycetes • Eukaryotic cells which are part of the kingdom “fungi” • Yeasts (S. cerevisiae, pichia pastoris) • non-filamentous • oval shape 5 – 8 um in diameter. • Rigid cell wall made of mannan (polymannose) • Usually divide asexually by budding • Most famous Saccharomyces cerevisiae(baker’s yeast – used in baking and brewing)

  9. Ascomycetes • “fungi” • Molds • filamentous – long, stringy, multicellular. • When growing on solids they form spore structures called conidia. • In submerged culture they grow as extended hyphae • Most common Aspergillus sps (industrial enzymes) and Penicillium sps(penicillin) • Trichoderma sps (not strictly a mold) is similar in morphology and behavior. (cellulases)

  10. Aspergillus

  11. Penicillium

  12. Other Eukaryotic Cells • Comparison between plant and animal cells

  13. Plant cells • Plant cells (Agrobacterium tumefaciens transfection system) • Usually grown as callus (solid surface) or suspension culture • Secondary metabolites (small molecules which are typically for defense response, but may have medical value • Production of flavors and scents • Transgenic plants

  14. Plant Cells • Plant Cell

  15. Insect cells • Insect cells (spodoptera frugiperda – caterpillar cells – baculovirus system) • High expression system • Infect S. frugiperda cells with genetically manipulated Autographa californica virus Up to 50% of intracellular protein, (much less if secreted)

  16. Mammalian cells • Production Cells • Chinese Hampster Ovary (cancer) cells • Hybridoma cells (fusion of B cells with immortal myeloma cancer cells) Used to produce monoclonal antibodies • Immunization of a mouse • Spleen removal from the mouse • Fusion of spleen cells with myeloma cells • Spleen cells are mortal but produce antibodies while myeloma cells are immortal but have no ability to produce asntibodies. • The resulting hybrid cell will have both of the above properties. • Screen the cell population for hybrid cells. • clone the isolated hybrid cells • Produce these cells in large quantities ( Scale-up ) • Tissue Culture – growth of specific organ tissues. (Stem cell technology)

  17. More Eukaryotic Cells • Mammalian Cell

  18. Hybridoma Cells

  19. Summary • There are a large variety of cells important for bioprocessing. • Source, production organism • Distinguish pro and eukaryotic cells • Size, growth rate, production levels, complexity of molecule, media formulation • Be familiar with names of certain yeast, mold, insect, mammalian, and plant cells • E. coli, Bacillus sps., S. cerevisiae, Pichia pastoris, various molds, Baculo virus (insect cells), CHO cells, hybridoma cells, plant cells

  20. Cell structures

  21. Cell Envelope • Influence processing conditions • Secretion systems • Filtration • Cell lysis • Media viscosity

  22. Generalized Bacterial Cell

  23. Cell Walls • Gram negative bacteria • Single layer of N-acetylglucosamine-muramic acid copolymer with a few tetrapeptide cross links. • O antigen, LPS endotoxin in cell wall • penicillin blocks cross linking • susceptible to lysozyme which cuts G-M bond • Gram positive bacteria • Multiple layer of N-AcG-M with many tetrapeptide cross links • penicillin blocks cross linking • susceptible to lysozyme which cuts G-M bond

  24. Gram positive vs Gram negative cell envelope

  25. E. Coli Cell Envelope

  26. E. Coli Cell Envelope

  27. E. Coli Cell Envelope

  28. Gram - Cell Wall

  29. Cell Walls • Molds • N-acetylglucosamine polymer (chitin) • Yeast • Glucosamine-mannan copolymer • Plants • Cellulose and hemicellulose with lignin and pectins

  30. Plant Cell wall

  31. Protoplasts • Protoplasts are intact cells with the cell wall removed • Increases ability to transfer genetic material into cells • Will regenerate cell wall if placed in appropriate media

  32. Protoplast

  33. Other Structures • Nucleoid • Bacteria have highly wound but simple chromosome that is much longer than the dimensions of the cell when unwound.

  34. Nucleoid

  35. E. Coli chromosome

  36. E. Coli Chromosome

  37. Eukaryotic Chromosomes • Eukaryotic chromosomes are complex structure which are highly organized to fit in the cell

  38. Eukaryotic Chromosome

  39. Protein Manufacturing Machinery • Ribosomes are small biological machines for protein manufacture • Attach one after another on m-RNA in bacterial cytosol as m-RNA is produced by RNA polymerase

  40. Ribosomes

  41. Polyribosomes

  42. Secretion Systems • Some ribosomes are membrane bound and aid in secretion • In gram negative cells, 90 % of proteins remain in cell, 10% make it through the membrane into the periplasm, and 1% (about 10) make it out of the cell through the outer membrane.

  43. Secretion in Gram - cells

  44. Protein secretion system • In mammalian cells: • Proteins are expressed through the ER membrane, transported to the Goli apparatus where they are post translationally packaged in vesicles. The vesicles diffuse to the cytoplasmic membrane where they fuse with the membrane and release their proteins to the extracellular space.

  45. Endoplasmic Reticulum

  46. Summary • The cell envelope is an important structure for bioprocessing • It determines how easily cells are broken (good and bad) • It controls antigenicity of the cell (endotoxins) • It determines antibiotic effectiveness (penicillin effectiveness) • It determines transformation efficiency (protoplasts) • It determines the ability of the cells to secrete proteins

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