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Digest remaining DNA with DNAse I 7 µl 10x RDD buffer 1 µl Superasin RNAse inhibitor

Digest remaining DNA with DNAse I 7 µl 10x RDD buffer 1 µl Superasin RNAse inhibitor 2.5 µl DNAse I Leave 30’ @ 37˚ C Add 15 µl 10 M ammonium acetate, then 85 µl isopropanol Leave >20’ @ -20˚ C Spin 10’ @16,000 g Decant supernatant, spin 10” then remove remainder with pipet

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Digest remaining DNA with DNAse I 7 µl 10x RDD buffer 1 µl Superasin RNAse inhibitor

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  1. Digest remaining DNA with DNAse I • 7 µl 10x RDD buffer • 1 µl Superasin RNAse inhibitor • 2.5 µl DNAse I • Leave 30’ @ 37˚ C • Add 15 µl 10 M ammonium acetate, then 85 µl isopropanol • Leave >20’ @ -20˚ C • Spin 10’ @16,000 g • Decant supernatant, spin 10” then remove remainder with pipet • Wash pellet with 100 µl 80% EtOH and spin 1’ @ 16000 • Carefully remove EtOH • Air dry with tube on side and cap open • Dissolve in 50µl mol. Grade water • Quantitate with nanodrop

  2. Prepare RNA mix • 1 µg RNA • 1 µl Random primer/poly dT mix

  3. Prepare RNA mix • 1 µg RNA • 1 µl Random primer/poly dT mix • Poly dT favors 3’ end, random hex favors 5’ end

  4. Prepare RNA mix in PCR tube • 1 µg RNA • 1 µl Random primer/poly dT mix • 1 µl 10 mM dNTP • Water to 12 µl • Leave 5’ @ 65˚ C, then chill to 4˚ C • Add • 4 µl 5x first strand buffer • 2 µl 100 mM DTT • 1 µl RNAse inhibitor • Leave > 2’ @ RT • Add 1 µl Superscript III • Leave 10’ @ 25 ˚ C, then 50’ @ 42 ˚ C • Inactivate by leaving 15’ @ 70˚ C • Use 1 µl for PCR with gene-specific primers

  5. Set up master mix for each primer combo on ice! • 2.5 µl 100x F primer (1 pMol/µl = 1µM final []) • 2.5 µl 100x R primer • 25 µl 10x PCR buffer • 5 µl 10 mM dNTP (200 µM final []) • 201 µl water • 1.5 µl Taq polymerase • Add 19 µl to 1 µl cDNA, and 19 µl to 1 µl genomic DNA • Run 30 cycles of 15” @ 94, 50-1”/cycle @ 50, 15” @ 72

  6. Plan A: Use plants to feed electrogenic bugs -> exude organics into rhizosphere

  7. General principle: Bacteria transfer e- from food to anode via direct contact, nanowires or a mediator. H+ diffuse to cathode to join e- forming H2O

  8. Geobacter species, Shewanella species In Geobacter sulfurreducens Om cytochromes transfer e- to anode via pili functioning as nanowires

  9. In Geobacter sulfurreducens Om cytochromes transfer e- to anode via pili functioning as nanowires 85% of the microorganisms consuming acetate in Fe(III)-reducing rice paddy soils were Geobacter species

  10. Geobacter metallireducens can oxidize ethanol but can’t use fumarate, Geobacter sulfurreducens can reduce fumarate but can’t use ethanol. Mixed cultures formed aggregates that oxidized ethanol & reduced fumarate. E- were transferred via pili & OmcS. Must be anaerobic!

  11. Many plant roots release ethanol upon hypoxia. Use them to feed Geobacter

  12. Many plant roots release ethanol upon hypoxia. Use them to feed Geobacter Overexpress OmcZ to enhance electron transfer

  13. Many plant roots release ethanol upon hypoxia. Use them to feed Geobacter Overexpress OmcZ to enhance electron transfer Make electrodes from graphite, Carbon cloth, gold or platinum

  14. Many plant roots release ethanol upon hypoxia. Use them to feed Geobacter Overexpress OmcZ to enhance electron transfer Make electrodes from graphite, Carbon cloth, gold or platinum Study role of pilin protein in electron transfer?

  15. Many plant roots release ethanol upon hypoxia. Use them to feed Geobacter Overexpress OmcZ to enhance electron transfer Make electrodes from graphite, Carbon cloth, gold or platinum Study role of pilin protein in electron transfer? Enhance organic exudation?

  16. Enhance organic exudation? Synechocystis sp. PCC 6803 ∆glgC secretes pyruvate when N-limited because it can’t make glycogen

  17. Many cyanobacteria reduce their surroundings in the light & make pili

  18. Green algae (Chlorella vulgaris, Dunaliella tertiolecta) or cyanobacteria (Synechocystis sp. PCC6803, Synechococcus sp.WH5701were used for bio-photovoltaics

  19. Green algae (Chlorella vulgaris, Dunaliella tertiolecta) or cyanobacteria (Synechocystis sp. PCC6803, Synechococcus sp.WH5701were used for bio-photovoltaics Study cyanobacterial pili? Express PilA? OmcZ?

  20. Engineering algae (or plants) to make H2

  21. Engineering algae (or plants) to make H2 Feed H2 to Geobacter?

  22. conversion of CO2 to ethylene (C2H4) in Synechocystis 6803 transformed with efe gene. Use ethylene to make plastics, diesel, gasoline, jet fuel or ethanol

  23. Changing Cyanobacteria to make a 5 carbon alcohol

  24. Botryococcus braunii partitions C from PS into sugar/fatty acid/terpenoid at ratios of 50 : 10 : 40 cf85 : 10 : 5 in most plants

  25. Light-independent (dark) reactions The Calvin cycle

  26. Light-independent (dark) reactions occur in the stroma of the chloroplast (pH 8) Consumes ATP & NADPH from light reactions regenerates ADP, Pi and NADP+

  27. Light-independent (dark) reactions Overall Reaction: 3 CO2 + 3 RuBP + 9 ATP + 6 NADPH = 3 RuBP + 9 ADP + 9 Pi + 6 NADP+ + 1 Glyceraldehyde 3-P

  28. Light-independent (dark) reactions 1) fixing CO2 2) reversing glycolysis 3) regenerating RuBP

  29. fixing CO2 1) RuBP binds CO2

  30. fixing CO2 • RuBP binds CO2 • 2) rapidly splits into two 3-Phosphoglycerate • therefore called C3 photosynthesis

  31. fixing CO2 • 1) CO2 is bound to RuBP • 2) rapidly splits into two 3-Phosphoglycerate • therefore called C3 photosynthesis • detected by immediately killing cells fed 14CO2

  32. fixing CO2 1) CO2 is bound to RuBP 2) rapidly splits into two 3-Phosphoglycerate 3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) the most important & abundant protein on earth

  33. fixing CO2 • 1) CO2 is bound to RuBP • 2) rapidly splits into two 3-Phosphoglycerate • 3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) • the most important & abundant protein on earth • Lousy Km

  34. fixing CO2 • 1) CO2 is bound to RuBP • 2) rapidly splits into two 3-Phosphoglycerate • 3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) • the most important & abundant protein on earth • Lousy Km • Rotten Vmax!

  35. Reversing glycolysis converts 3-Phosphoglycerate to G3P consumes 1 ATP & 1 NADPH

  36. Reversing glycolysis • G3P has 2 possible fates • 1) 1 in 6 becomes (CH2O)n

  37. Reversing glycolysis • G3P has 2 possible fates • 1) 1 in 6 becomes (CH2O)n • 2) 5 in 6 regenerate RuBP

  38. Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell)

  39. Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose

  40. Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose Pi/triosePO4 antiporter only trades DHAP for Pi

  41. Reversing glycolysis 1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell) or is converted to DHAP & exported to cytoplasm to make sucrose Pi/triosePO4 antiporter only trades DHAP for Pi mechanism to regulate PS

  42. Regenerating RuBP • G3P has 2 possible fates • 5 in 6 regenerate RuBP • necessary to keep cycle going

  43. Regenerating RuBP Basic problem: converting a 3C to a 5C compound feed in five3C sugars, recover three5C sugars

  44. Regenerating RuBP Basic problem: converting a 3C to a 5C compound must assemble intermediates that can be broken into 5 C sugars after adding 3C subunit

  45. Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C

  46. Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C 4C + 3C = 7C

  47. Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C 4C + 3C = 7C 7C + 3C = 5C + 5C

  48. Regenerating RuBP making intermediates that can be broken into 5 C sugars after adding 3C subunits 3C + 3C + 3C = 5C + 4C 4C + 3C = 7C 7C + 3C = 5C + 5C Uses 1 ATP/RuBP

  49. Light-independent (dark) reactions build up pools of intermediates , occasionally remove one very complicated book-keeping

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