Digest remaining DNA with DNAse I

1

1. Digest remaining DNA with DNAse I
2. 7 µl 10x RDD buffer
3. 1 µl Superasin RNAse inhibitor
4. 2.5 µl DNAse I
5. Leave 30 _at_ 37 C
6. Add 15 µl 10 M ammonium acetate, then 85 µl
   isopropanol
7. Leave gt20 _at_ -20 C
8. Spin 10 _at_16,000 g
9. Decant supernatant, spin 10 then remove
   remainder with pipet
10. Wash pellet with 100 µl 80 EtOH and spin 1 _at_
    16000
11. Carefully remove EtOH
12. Air dry with tube on side and cap open
13. Dissolve in 50µl mol. Grade water
14. Quantitate with nanodrop

2

1. Prepare RNA mix
2. 1 µg RNA
3. 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

1. Prepare RNA mix in PCR tube
2. 1 µg RNA
3. 1 µl Random primer/poly dT mix
4. 1 µl 10 mM dNTP
5. Water to 12 µl
6. Leave 5 _at_ 65 C, then chill to 4 C
7. Add
8. 4 µl 5x first strand buffer
9. 2 µl 100 mM DTT
10. 1 µl RNAse inhibitor
11. Leave gt 2 _at_ RT
12. Add 1 µl Superscript III
13. Leave 10 _at_ 25 C, then 50 _at_ 42 C
14. Inactivate by leaving 15 _at_ 70 C
15. Use 1 µl for PCR with gene-specific primers

5

1. Set up master mix for each primer combo on ice!
2. 2.5 µl 100x F primer (1 pMol/µl 1µM final )
3. 2.5 µl 100x R primer
4. 25 µl 10x PCR buffer
5. 5 µl 10 mM dNTP (200 µM final )
6. 201 µl water
7. 1.5 µl Taq polymerase
8. Add 19 µl to 1 µl cDNA, and 19 µl to 1 µl genomic
   DNA
9. Run 30 cycles of 15 _at_ 94, 50-1/cycle _at_ 50, 15
   _at_ 72

6
Plan A Use plants to feed electrogenic bugs -gt
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
cant use fumarate, Geobacter sulfurreducens can
reduce fumarate but cant 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 cant 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
(No Transcript)
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 gt 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
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
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 cant
64
PHOTORESPIRATION Rubisco can use O2 as substrate
instead of CO2 RuBP O2 ltgt 3-phosphoglycerate
phosphoglycolate
65
PHOTORESPIRATION Rubisco can use O2 as substrate
instead of CO2 RuBP O2 ltgt 3-phosphoglycerate
Phosphoglycolate Releases CO2 without making ATP
or NADH
66
PHOTORESPIRATION Releases CO2 without making ATP
or NADH Called photorespiration undoes
photosynthesis
67
PHOTORESPIRATION Rubisco can use O2 as substrate
instead of CO2 RuBP O2 ltgt 3-phosphoglycerate
Phosphoglycolate C3 plants can lose 25-50 of
their fixed carbon
68
PHOTORESPIRATION Rubisco can use O2 as substrate
instead of CO2 RuBP O2 ltgt 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!