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

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

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

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

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

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

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

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

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

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!

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

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

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

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?

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?

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

Many cyanobacteria reduce their surroundings in the light & make pili

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

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

Engineering algae (or plants) to make H2

Engineering algae (or plants) to make H2

Feed H2 to Geobacter?

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

Changing Cyanobacteria to make a 5 carbon alcohol

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

Light-independent (dark) reactionsThe Calvin cycle

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

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

Light-independent (dark) reactions1) fixing CO2

2) reversing glycolysis3) regenerating RuBP

fixing CO2

1) RuBP binds CO2

fixing CO2

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

• therefore called C3 photosynthesis

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

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

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

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

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!

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

Reversing glycolysisG3P has 2 possible fates

1) 1 in 6 becomes (CH2O)n

Reversing glycolysisG3P has 2 possible fates

1) 1 in 6 becomes (CH2O)n

2) 5 in 6 regenerate RuBP

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

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

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

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

Regenerating RuBPG3P has 2 possible fates

5 in 6 regenerate RuBPnecessary to keep cycle going

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

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

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

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

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

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

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

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

Regulating the Calvin CycleRubisco is main rate-limiting step

Regulating the Calvin CycleRubisco is main rate-limiting step

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

Regulating the Calvin CycleRubisco is main rate-limiting step

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

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)

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

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

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

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+]

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

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

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

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

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

RuBP + O2 <=> 3-phosphoglycerate + phosphoglycolate

PHOTORESPIRATION Rubisco can use O2 as substrate instead of CO2

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

PHOTORESPIRATION Releases CO2 without making ATP or NADH

Called photorespiration : undoes photosynthesis

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

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

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