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1. Digest remaining DNA with DNAse I 1. 7 µl 10x RDD buffer 2. 1 µl Superasin RNAse inhibitor 3. 2.5 µl DNAse I 2. Leave 30’ @ 37˚ C 3. Add 15 µl 10 M ammonium acetate, then 85 µl isopropanol 4. Leave >20’ @ -20˚ C 5. Spin 10’ @16,000 g 6. Decant supernatant, spin 10” then remove remainder with pipet 7. Wash pellet with 100 µl 80% EtOH and spin 1’ @ 16000 8. Carefully remove EtOH 9. Air dry with tube on side and cap open 10.Dissolve in 50µl mol. Grade water 11.Quantitate with nanodrop
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1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Jan 01, 2016

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Candace King

use ethanol

enhanceelectron transfer

use fumarate

bacteria transfer e

use plants

geobacteroverexpress

random primerpoly dt

carbon cloth

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Page 1: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

1. Digest remaining DNA with DNAse I1. 7 µl 10x RDD buffer2. 1 µl Superasin RNAse inhibitor3. 2.5 µl DNAse I

2. Leave 30’ @ 37˚ C3. Add 15 µl 10 M ammonium acetate, then 85 µl

isopropanol4. Leave >20’ @ -20˚ C5. Spin 10’ @16,000 g6. Decant supernatant, spin 10” then remove remainder

with pipet7. Wash pellet with 100 µl 80% EtOH and spin 1’ @

160008. Carefully remove EtOH9. Air dry with tube on side and cap open10.Dissolve in 50µl mol. Grade water11.Quantitate with nanodrop

Page 2: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

1. Prepare RNA mix1. 1 µg RNA2. 1 µl Random primer/poly dT mix

Page 3: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

1. Prepare RNA mix1. 1 µg RNA2. 1 µl Random primer/poly dT mix• Poly dT favors 3’ end, random hex favors 5’ end

Page 4: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

1. Prepare RNA mix in PCR tube1. 1 µg RNA2. 1 µl Random primer/poly dT mix3. 1 µl 10 mM dNTP4. Water to 12 µl

2. Leave 5’ @ 65˚ C, then chill to 4˚ C3. Add

1. 4 µl 5x first strand buffer2. 2 µl 100 mM DTT3. 1 µl RNAse inhibitor

4. Leave > 2’ @ RT5. Add 1 µl Superscript III6. Leave 10’ @ 25 ˚ C, then 50’ @ 42 ˚ C7. Inactivate by leaving 15’ @ 70˚ C8. Use 1 µl for PCR with gene-specific primers

Page 5: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

1. Set up master mix for each primer combo on ice!1. 2.5 µl 100x F primer (1 pMol/µl = 1µM final [])2. 2.5 µl 100x R primer3. 25 µl 10x PCR buffer4. 5 µl 10 mM dNTP (200 µM final [])5. 201 µl water6. 1.5 µl Taq polymerase

2. Add 19 µl to 1 µl cDNA, and 19 µl to 1 µl genomic DNA3. Run 30 cycles of 15” @ 94, 50-1”/cycle @ 50, 15” @ 72

Page 6: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 7: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 8: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 9: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

In Geobacter sulfurreducens Om cytochromes transfer e- to anode via pili functioning as nanowires85% of the microorganisms consuming acetate in Fe(III)-reducing rice paddy soils were Geobacter species

Page 10: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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!

Page 11: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 12: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Many plant roots release ethanol upon hypoxia.Use them to feed GeobacterOverexpress OmcZ to enhanceelectron transfer

Page 13: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Many plant roots release ethanol upon hypoxia.Use them to feed GeobacterOverexpress OmcZ to enhanceelectron transfer Make electrodes from graphite,Carbon cloth, gold or platinum

Page 14: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Many plant roots release ethanol upon hypoxia.Use them to feed GeobacterOverexpress OmcZ to enhanceelectron transfer Make electrodes from graphite,Carbon cloth, gold or platinumStudy role of pilin protein in electron transfer?

Page 15: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Many plant roots release ethanol upon hypoxia.Use them to feed GeobacterOverexpress OmcZ to enhanceelectron transfer Make electrodes from graphite,Carbon cloth, gold or platinumStudy role of pilin protein in electron transfer?Enhance organic exudation?

Page 16: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 17: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Many cyanobacteria reduce their surroundings in the light & make pili

Page 18: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 19: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 20: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.
Page 21: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Engineering algae (or plants) to make H2

Page 22: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Engineering algae (or plants) to make H2

Feed H2 to Geobacter?

Page 23: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 24: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Changing Cyanobacteria to make a 5 carbon alcohol

Page 25: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 26: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Light-independent (dark) reactionsThe Calvin cycle

Page 27: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 28: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 29: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Light-independent (dark) reactions1) fixing CO2

2) reversing glycolysis3) regenerating RuBP

Page 30: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

fixing CO2

1) RuBP binds CO2

Page 31: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

fixing CO2

1) RuBP binds CO2 2) rapidly splits into two 3-Phosphoglycerate

• therefore called C3 photosynthesis

Page 32: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate

• therefore called C3 photosynthesis•detected by immediately killing cells fed 14CO2

Page 33: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase)the most important & abundant protein on earth

Page 34: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase)the most important & abundant protein on earth

•Lousy Km

Page 35: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

fixing CO21) CO2 is bound to RuBP2) rapidly splits into two 3-Phosphoglycerate3) catalyzed by Rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase)the most important & abundant protein on earth

•Lousy Km

•Rotten Vmax!

Page 36: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Reversing glycolysisconverts 3-Phosphoglycerate to G3Pconsumes 1 ATP & 1 NADPH

Page 37: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Reversing glycolysisG3P has 2 possible fates

1) 1 in 6 becomes (CH2O)n

Page 38: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Reversing glycolysisG3P has 2 possible fates

1) 1 in 6 becomes (CH2O)n

2) 5 in 6 regenerate RuBP

Page 39: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Reversing glycolysis1 in 6 G3P becomes (CH2O)n either becomes starch in chloroplast (to store in cell)

Page 40: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Reversing glycolysis1 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

Page 41: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Reversing glycolysis1 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 sucrosePi/triosePO4

antiporter onlytrades DHAP for Pi

Page 42: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Reversing glycolysis1 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 sucrosePi/triosePO4

antiporter onlytrades DHAP for Pimechanism to regulate PS

Page 43: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regenerating RuBPG3P has 2 possible fates

5 in 6 regenerate RuBPnecessary to keep cycle going

Page 44: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regenerating RuBPBasic problem: converting a 3C to a 5C compoundfeed in five 3C sugars, recover three 5C sugars

Page 45: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regenerating RuBPBasic problem: converting a 3C to a 5C compoundmust assemble intermediates that can be broken into 5 C sugars after adding 3C subunit

Page 46: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C

Page 47: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C4C + 3C = 7C

Page 48: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C4C + 3C = 7C7C + 3C = 5C + 5C

Page 49: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regenerating RuBPmaking intermediates that can be broken into 5 C sugars after adding 3C subunits3C + 3C + 3C = 5C + 4C4C + 3C = 7C7C + 3C = 5C + 5CUses 1 ATP/RuBP

Page 50: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Light-independent (dark) reactionsbuild up pools of intermediates , occasionally remove onevery complicated book-keeping

Page 51: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Light-independent (dark) reactionsbuild up pools of intermediates , occasionally remove onevery complicated book-keepingUse 12 NADPH and 18 ATP to make one 6C sugar

Page 52: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

Page 53: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways 1) Rubisco activase : uses ATP to activate rubisco

Page 54: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

Page 55: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pH: rubisco is most active at pH > 8 (in dark pH is ~7.2)

Page 56: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pHb) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in dark

Page 57: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pHb) [Mg2+]: in light [Mg2+] in stroma is ~ 10x greater than in darkMg2+ moves from thylakoid lumen to stroma to maintain charge neutrality

Page 58: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

a) pHb) [Mg2+]c) CO2 is an allosteric activator of rubisco that only binds at high pH and high [Mg2+]also: stomates open in the light

Page 59: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleRubisco is main rate-limiting step

indirectly regulated by light 2 ways1) Rubisco activase2) Light-induced changes in stroma

Several other Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also activated by high pH & [Mg2+]

Page 60: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleSeveral Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin

contain disulfide bonds which get oxidized in the dark

Page 61: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleSeveral Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin

contain disulfide bonds which get oxidized in the darkin light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzyme

S - S

2Fdox

2Fdred

PSI +

PSII

light

2e-

oxidizedthioredoxin

reducedthioredoxin

S - S

SH SHoxidizedenzyme

(inactive)

reducedenzyme(active)

SH SH

Page 62: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleSeveral Calvin cycle enzymes (e.g.Fructose-1,6-bisphosphatase) are also regulated by thioredoxin

contain disulfide bonds which get oxidized in the darkin light, ferredoxin reduces thioredoxin, thioredoxin reduces these disulfide bonds to activate the enzymeHow light reactions talk to the Calvin cycle

S - S

2Fdox

2Fdred

PSI +

PSII

light

2e-

oxidizedthioredoxin

reducedthioredoxin

S - S

SH SHoxidizedenzyme

(inactive)

reducedenzyme(active)

SH SH

Page 63: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

Regulating the Calvin CycleOverall = enzyme synthesisMost encoded by nucleusRbcS = nucleusRbcL = CP[rbcS] regulates translation of mRNA for rbcL!Plastids also signal nucleus: GUN mutants can’t

Page 64: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate

Page 65: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

RuBP + O2 <=> 3-phosphoglycerate + PhosphoglycolateReleases CO2 without making ATP or NADH

Page 66: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

PHOTORESPIRATION Releases CO2 without making ATP or NADH

Called photorespiration : undoes photosynthesis

Page 67: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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

Page 68: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

RuBP + O2 <=> 3-phosphoglycerate + PhosphoglycolateC3 plants can lose 25%-50% of their fixed carbonBoth rxns occur at same active site

Page 69: 1.Digest remaining DNA with DNAse I 1.7 µl 10x RDD buffer 2.1 µl Superasin RNAse inhibitor 3.2.5 µl DNAse I 2.Leave 30’ @ 37˚ C 3.Add 15 µl 10 M ammonium.

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


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articles.pdf · Temperature stability to -1960C, autoclavable at 121 oc (2 bar), acc. DIN EN 285. Gamma-ray sterilized (SAL 10-6 ), RNase-, DNase-, DNA- and endotoxin-free. Large

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IntelliPlex 1000 Resource... · 2020. 9. 18. · Introduction_P-1_V01 Sep-2018 - Page 6 Buffer Bottles Distilled Water (1) •Use ultrapure DNase/RNase-free distilled water Washing

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PCR Products · • Available in ultra-clear, assorted, or specific colors. • Certified free of detectable RNase, DNase, DNA, PCR inhibitors, and tested pyrogen-free. IsoFreeze

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pathogenic subtypesPCRmax Ltd qPCR test TM Aspergillus all · 2018-03-01 · Aspergillus_spp primer/probe mix (BROWN) 1 µl Final Volume 15 µl 10 µl RNase/DNase free water (WHITE)

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Company profile temperature storage system.pdf · DNase and RNase free and non-pyrogenic using the highest quality medical-grade plastic ... System-Refrigerators & Freezers CryoKING

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PapilloCheck - GBO · Steril, DNase içermeyen mikropipet filtre ucu4 0,5 µL - 10 µL 0,5 µL - 20 µL 10 µL - 100µL 10µL - 200 µL 100 µL - 1000 µL 765 288 774 288 772 288

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Challenges and Controls in Metagenomics · 2015-09-06 · Collect samples in Sterile DNase, RNase, DNA, and RNA-free I. Lugol’s: Transfer 50 mls of each sample to sterile 50 ml

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Round Bottom Culture Tubes Conical Centrifuge Tubes High ... Bio/Centrifuge Tube... · Printed Graduation & Marking Spot Sterile; RNase & DNase Free Culture Tubes (suppbed With dual

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Products Catalog - Catalog - CE Version - P134 - Rev 5.0.d. · Supermix, RT Mix (RNA Assays Only), Positive Controls, Internal Control, RNase/DNase Free Water Reportable Results:

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