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Digest remaining DNA with DNAse I

Published byCandace King Modified over 9 years ago

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Presentation on theme: "Digest remaining DNA with DNAse I"— Presentation transcript:

1 Digest remaining DNA with DNAse I
7 µl 10x RDD buffer 1 µl Superasin RNAse inhibitor 2.5 µl DNAse I Leave 37˚ C Add 15 µl 10 M ammonium acetate, then 85 µl isopropanol Leave -20˚ C Spin g Decant supernatant, spin 10” then remove remainder with pipet Wash pellet with 100 µl 80% EtOH and spin 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 65˚ C, then chill to 4˚ C Add 4 µl 5x first strand buffer 2 µl 100 mM DTT 1 µl RNAse inhibitor Leave > RT Add 1 µl Superscript III Leave 25 ˚ C, then 42 ˚ C Inactivate by leaving 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 94, 50, 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

21 Engineering algae (or plants) to make H2

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

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

24 Changing Cyanobacteria to make a 5 carbon alcohol

25 Botryococcus braunii partitions C from PS into sugar/fatty acid/terpenoid at ratios of 50 : 10 : 40 cf 85 : 10 : 5 in most plants

26 Light-independent (dark) reactions
The Calvin cycle

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

28 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

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

30 fixing CO2 1) RuBP binds CO2

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

32 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

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

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

35 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!

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

37 Reversing glycolysis G3P has 2 possible fates 1) 1 in 6 becomes (CH2O)n

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

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

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

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

42 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

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

44 Regenerating RuBP Basic problem: converting a 3C to a 5C compound feed in five 3C sugars, recover three 5C sugars

45 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

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

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

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

49 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

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

51 Light-independent (dark) reactions
build up pools of intermediates , occasionally remove one very complicated book-keeping Use 12 NADPH and 18 ATP to make one 6C sugar

52 Regulating the Calvin Cycle
Rubisco is main rate-limiting step

53 Regulating the Calvin Cycle
Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase : uses ATP to activate rubisco

54 Regulating the Calvin Cycle
Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma

55 Regulating the Calvin Cycle
Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH: rubisco is most active at pH > 8 (in dark pH is ~7.2)

56 Regulating the Calvin Cycle
Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark

57 Regulating the Calvin Cycle
Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark Mg2+ moves from thylakoid lumen to stroma to maintain charge neutrality

58 Regulating the Calvin Cycle
Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma a) pH b) [Mg2+] c) CO2 is an allosteric activator of rubisco that only binds at high pH and high [Mg2+] also: stomates open in the light

59 Regulating the Calvin Cycle
Rubisco is main rate-limiting step indirectly regulated by light 2 ways 1) Rubisco activase 2) Light-induced changes in stroma Several other Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also activated by high pH & [Mg2+]

60 Regulating the Calvin Cycle
Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark

61 Regulating the Calvin Cycle
Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark in light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme S - S 2Fdox 2Fdred PSI + PSII light 2e- oxidized thioredoxin reduced SH enzyme (inactive) (active)

62 Regulating the Calvin Cycle
Several Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin contain disulfide bonds which get oxidized in the dark in light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme How light reactions talk to the Calvin cycle S - S 2Fdox 2Fdred PSI + PSII light 2e- oxidized thioredoxin reduced SH enzyme (inactive) (active)

63 Regulating the Calvin Cycle
Overall = enzyme synthesis Most encoded by nucleus RbcS = nucleus RbcL = CP [rbcS] regulates translation of mRNA for rbcL! Plastids also signal nucleus: GUN mutants can’t

64 RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate
PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate

65 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate
PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate Releases CO2 without making ATP or NADH

66 PHOTORESPIRATION Releases CO2 without making ATP or NADH Called photorespiration : undoes photosynthesis

67 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate
PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon

68 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate
PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2 RuBP + O2 <=> 3-phosphoglycerate + Phosphoglycolate C3 plants can lose 25%-50% of their fixed carbon Both rxns occur at same active site

69 PHOTORESPIRATION C3 plants can lose 25%-50% of their fixed carbon phosphoglycolate is converted to glycolate : poison!

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