Bio 226: Cell and Molecular Biology - Wilkes University

Bio 226: Cell and Molecular Biology - Wilkes University

Common themes 1.Nutrient deprivation N and biodiesel N and H2 production S and biodiesel Biotin and biodiesel 2. Hydrogen production

N deprivation Knock down hydrogenases Knock up hydrogen synthases, H+ pumps 3.Gene knockouts FFA recycling H2 metabolism N metabolism Cell walls

Abiotic stresses Salinity osmotic Temperature Common themes 1.Growth in different media Differ in [N] or other nutrients

2.Growth in common medium, then change Harvest, then resuspend in new media Add something to medium Salt Biotin/avidin Inducer Inhibitor

Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Hypothesis: manipulating the internal environment of

cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth

Predictions? Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions? Growth?

Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions? Growth? Photosynthesis?

Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions? Growth? Photosynthesis?

Respiration? Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions? Growth?

Photosynthesis? Respiration? Protein synthesis? Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth

Predictions? Growth? Photosynthesis? Respiration? Protein synthesis? DNA synthesis? Hypothesis: manipulating the internal environment of

cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions? Growth? Photosynthesis? Respiration? Protein synthesis?

DNA synthesis? Lipid synthesis? Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions?

Growth? Photosynthesis? Respiration? Protein synthesis? DNA synthesis? Lipid synthesis? Hydrogen synthesis?

Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions? Growth? Photosynthesis? Respiration?

Protein synthesis? DNA synthesis? Lipid synthesis? Hydrogen synthesis? How to test? Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production

Will change allocation of photosynthate Will invest their income in energy stores cf growth Predictions? Growth? Photosynthesis? Respiration? Protein synthesis? DNA synthesis?

Lipid synthesis? Hydrogen synthesis? How to test? Alter external conditions Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate

Will invest their income in energy stores cf growth Predictions? Growth? Photosynthesis? Respiration? Protein synthesis? DNA synthesis? Lipid synthesis?

Hydrogen synthesis? How to test? Alter external conditions Alter internal conditions by bioengineering Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate

Will invest their income in energy stores cf growth Predictions? Growth? Photosynthesis? Respiration? Protein synthesis? DNA synthesis? Lipid synthesis?

Hydrogen synthesis? How to test? Alter external conditions Alter internal conditions by bioengineering Then measure growth, physiology and biofuels Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production

Alter external conditions Nutrients N, S, P, cofactors (including biotin) Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Alter external conditions Nutrients

N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Alter external conditions Nutrients

N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Alter external conditions

Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium Temperature Hypothesis: manipulating the internal environment of

cyanobacteria will affect biofuel production Alter external conditions Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium Temperature

Light: intensity & duration Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Alter external conditions Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential

NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium Temperature Light: intensity & duration Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Alter external conditions

Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium Temperature Light: intensity & duration Turn down light reactions with atrazine

Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Alter external conditions Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG

pCO2: pCO2 in air and [HCO3-] in medium Temperature Light: intensity & duration Turn down light reactions with atrazine Bioengineer internal changes Nutrients (including HCO3-) by altering transporters Hypothesis: manipulating the internal environment of

cyanobacteria will affect biofuel production Alter external conditions Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium Temperature

Light: intensity & duration Turn down light reactions with atrazine Bioengineer internal changes Nutrients (including HCO3-) by altering transporters Light reactions Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production

Alter external conditions Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium Temperature Light: intensity & duration

Turn down light reactions with atrazine Bioengineer internal changes Nutrients (including HCO3-) by altering transporters Light reactions H2 production via N2ases, H2ases, H+ pumps, etc Redirect photosynthate K/O FFA recycling

Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Alter external conditions Nutrients N, S, P, cofactors (including biotin) Salinity/ water potential NaCl vs KCl vs mannitol/sorbitol/PEG pCO2: pCO2 in air and [HCO3-] in medium

Temperature Light: intensity & duration Turn down light reactions with atrazine Bioengineer internal changes Nutrients (including HCO3-) by altering transporters Light reactions H2 production via N2ases, H2ases, H+ pumps, etc Redirect photosynthate

K/O FFA recycling Suggested Game Plan 1.Run everything in parallel in Synechococcus elongatus and Anabaena We have experience growing S. elongatus + expertise & materials to engineer its genome Anabaena will be the exptl organism

Suggested Game Plan 1.Run everything in parallel in Synechococcus elongatus and Anabaena We have experience growing S. elongatus + expertise & materials to engineer its genome Anabaena will be the exptl organism 2. For environmental folks:

Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions Suggested Game Plan 1.Run everything in parallel in Synechococcus elongatus and Anabaena We have experience growing S. elongatus + expertise & materials to engineer its genome

Anabaena will be the exptl organism 2. For environmental folks: Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions At suitable intervals measure Growth Heterocysts H2 production

Photosynthesis Respiration DNA, RNA, gene expression in general Lipids Suggested Game Plan 2. For environmental folks: Grow large batches of S. elongatus and Anabaena, then

subdivide into different media/ conditions At suitable intervals measure Growth Heterocysts H2 production Photosynthesis Respiration DNA, RNA, gene expression in general

Lipids 3. For gene folks Identify genes predicted to affect biofuel production Suggested Game Plan 2. For environmental folks: Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions

At suitable intervals measure Growth Heterocysts H2 production Photosynthesis Respiration DNA, RNA, gene expression in general Lipids

3. For gene folks Identify genes predicted to affect biofuel production Nutrient uptake or metabolism Suggested Game Plan 2. For environmental folks: Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions

At suitable intervals measure Growth Heterocysts H2 production Photosynthesis Respiration DNA, RNA, gene expression in general Lipids

3. For gene folks Identify genes predicted to affect biofuel production Nutrient uptake or metabolism Lipid unsaturation Suggested Game Plan 2. For environmental folks: Grow large batches of S. elongatus and Anabaena, then

subdivide into different media/ conditions At suitable intervals measure Growth Heterocysts H2 production Photosynthesis Respiration DNA, RNA, gene expression in general

Lipids 3. For gene folks Identify genes predicted to affect biofuel production Nutrient uptake or metabolism Lipid unsaturation Alternate biofuels Suggested Game Plan

3. For gene folks Identify genes predicted to affect biofuel production Nutrient uptake or metabolism Lipid unsaturation Alternate biofuels 4. Clone and transform genes into host

Suggested Game Plan 3. For gene folks Identify genes predicted to affect biofuel production Nutrient uptake or metabolism Lipid unsaturation Alternate biofuels 4. Clone and transform genes into host

5. Measure effects of transgenes on physiology and biofuel production Suggested Game Plan 3. For gene folks Identify genes predicted to affect biofuel production Nutrient uptake or metabolism

Lipid unsaturation Alternate biofuels 4. Clone and transform genes into host 5. Measure effects of transgenes on physiology and biofuel production Monday Environmental folks make the various media and start growing cells

Suggested Game Plan 3. For gene folks Identify genes predicted to affect biofuel production Nutrient uptake or metabolism Lipid unsaturation Alternate biofuels

4. Clone and transform genes into host 5. Measure effects of transgenes on physiology and biofuel production Monday Environmental folks make the various media and start growing cells Gene folks work on identifying suitable targets and devising strategies to clone them.

Mineral Nutrition Soil nutrients Amounts & availability vary Many are immobile, eg P, Fe Mineral Nutrition Immobile nutrients must be mined

Root hairs get close Mycorrhizae get closer Rhizosphere Endomycorrhizae invade root cells: Vesicular/Arbuscular Most angiosperms, especially in nutrient-poor soils Deliver nutrients into symplast or release them when arbuscule dies

Also find bacteria, actinomycetes, protozoa associated with root surface = rhizosphere Rhizosphere Also find bacteria, actinomycetes, protozoa associated with root surface = rhizosphere Plants feed them lots of C! They help make nutrients available

N-fixing bacteria supply N to many plant spp Nutrient uptake Most nutrients are dissolved in water Nutrient uptake Most nutrients are dissolved in water Enter root through apoplast until hit endodermis

Nutrient uptake Most nutrients are dissolved in water Enter root through apoplast until hit endodermis Then must cross plasma membrane Crossing membranes A) Diffusion through bilayer

B) Difusion through protein pore Selective C) Facilitated diffusion D) Active transport E) Bulk transport Active 1) Exocytosis 2) Endocytosis

Nutrient uptake Then must cross plasma membrane Gases, small uncharged & non-polar molecules diffuse Nutrient uptake Then must cross plasma membrane Gases, small uncharged & non-polar molecules diffuse

down their [ ] Important for CO2, auxin & NH3 transport Nutrient uptake Then must cross plasma membrane Gases, small uncharged & non-polar molecules diffuse down their [ ] Polar chems must go through proteins!

Selective Transport 1) Channels integral membrane proteins with pore that specific ions diffuse through Selective Transport 1) Channels

integral membrane proteins with pore that specific ions diffuse through depends on size & charge Channels integral membrane proteins with pore that specific ions diffuse through

depends on size & charge O in selectivity filter bind ion (replace H2O) Channels integral membrane proteins with pore that specific ions diffuse through depends on size & charge

O in selectivity filter bind ion (replace H2O) only right one fits Channels O in selectivity filter bind ion (replace H2O) only right one fits

driving force? electrochemical Channels driving force : electrochemical non-saturable Channels

driving force : electrochemical non-saturable regulate by opening & closing Channels regulate by opening & closing ligand-gated channels open/close when bind specific chemicals

Channels ligand-gated channels open/close when bind specific chemicals Stress-activated channels open/close in response to mechanical stimulation Channels

Stress-activated channels open/close in response to mechanical stimulation voltage-gated channels open/close in response to changes in electrical potential Channels

Old model: S4 slides up/down Paddle model: S4 rotates Channels Old model: S4 slides up/down Paddle model: S4 rotates 3 states 1.Closed

2.Open 3. Inactivated Selective Transport 1) Channels 2) Facilitated Diffusion (carriers) Carrier binds molecule

Selective Transport Facilitated Diffusion (carriers) Carrier binds molecule carries it through membrane & releases it inside Selective Transport Facilitated Diffusion (carriers)

Carrier binds molecule carries it through membrane & releases it inside driving force = [ ] Selective Transport Facilitated Diffusion (carriers) Carrier binds molecule

carries it through membrane & releases it inside driving force = [ ] Important for sugar transport Selective Transport Facilitated Diffusion (carriers)

Characteristics 1) saturable 2) specific 3) passive: transports down [] Selective Transport 1) Channels

2) Facilitated Diffusion (carriers) Passive transport should equalize [ ] Nothing in a plant cell is at equilibrium! Selective Transport Passive transport should equalize [ ] Nothing in a plant cell is at equilibrium! Solution: use energy to transport specific ions against

their [ ] Active Transport Integral membrane proteins use energy to transport specific ions against their [ ] allow cells to concentrate some chemicals, exclude others Active Transport

Characteristics 1) saturable 105-106 ions/s 102-104 molecules/s Active Transport

Characteristics 1) saturable 2) specific Active Transport Characteristics 1) saturable 2) specific

3) active: transport up [ ] (or Em) 4 classes of Active transport ATPase proteins 1) P-type ATPases (P = phosphorylation) Na/K pump Ca pump in ER & PM H+ pump in PM pumps H+ out of cell

4 classes of Active transport ATPase proteins 1) P-type ATPases (P = phosphorylation) 2) V-type ATPases (V = vacuole) H+ pump in vacuoles 4 classes of Active transport ATPase proteins 1) P-type ATPases (P = phosphorylation)

2) V-type ATPases (V=vacuole) 3) F-type ATPases (F = factor) a.k.a. ATP synthases mitochondrial ATP synthase chloroplast ATP synthase 4 classes of Active transport ATPase proteins 1) P-type ATPases (P = phosphorylation) 2) V-type ATPases (V = vacuole)

3) F-type ATPases (F = factor) 4) ABC ATPases (ABC = ATP Binding Cassette) multidrug resistance proteins 4 classes of Active transport ATPase proteins 1) P-type ATPases (P = phosphorylation) 2) V-type ATPases (V = vacuole) 3) F-type ATPases (F = factor)

4) ABC ATPases (ABC = ATP Binding Cassette) multidrug resistance proteins pump hydrophobic drugs out of cells very broad specificity Secondary active transport Uses [ ] created by active transport to pump something else across a membrane against its [ ]

Secondary active transport Uses [ ] created by active transport to pump something else across a membrane against its [ ] Symport: both substances pumped same way Secondary active transport Uses [ ] created by active transport to pump something

else across a membrane against its [ ] Symport: both substances pumped same way Antiport: substances pumped opposite ways Secondary active transport Uses [ ] created by active transport to pump something else across a membrane against its [ ]

Symport: both substances pumped same way Antiport: substances pumped opposite ways Nutrient uptake Gases enter/exit by diffusion down their [ ] Ions vary dramatically!

Nutrient uptake Ions vary dramatically! H+ is actively pumped out of cell by P-type H+ -ATPase Nutrient uptake Ions vary dramatically H+ is actively pumped out of cell by P-type H+ -ATPase

and into vacuole by V-type ATPase & PPase Nutrient uptake H+ is actively pumped out of cell by P-type H+ -ATPase and into vacuole by V-type ATPase & PPase Main way plants make membrane potential (Em)! Nutrient uptake

H+ is actively pumped out of cell by P-type H+ -ATPase and into vacuole by V-type ATPase & PPase Main way plants make membrane potential (Em)! Used for many kinds of transport! Nutrient uptake Many ions are imported by multiple transporters with varying affinities

Nutrient uptake Many ions are imported by multiple transporters with varying affinities K+ diffuses through channels down Em: low affinity Nutrient uptake Many ions are imported by multiple transporters with

varying affinities K+ diffuses through channels down Em: low affinity Also taken up by H+ symporters : high affinity Nutrient uptake Many ions are imported by multiple transporters with varying affinities K+ diffuses through channels down Em: low affinity

Also taken up by H+ symporters : high affinity Low affinity is cheaper but less effective Nutrient uptake K+ diffuses through channels down Em: low affinity Also taken up by H+ symporters : high affinity Low affinity is cheaper but less effective

some channels also transport Na+ Nutrient uptake K+ diffuses through channels down Em: low affinity Also taken up by H+ symporters : high affinity Low affinity is cheaper but less effective some channels also transport Na+ why Na+ slows K+ uptake?

Nutrient uptake K+ diffuses through channels down Em: low affinity Also taken up by H+ symporters : high affinity Low affinity is cheaper but less effective some channels also transport Na+ why Na+ slows K+ uptake? Na+ is also expelled

by H+ antiport Nutrient uptake Ca2+ is expelled by P-type ATPases in PM Nutrient uptake Ca2+ is expelled by P-type ATPases in PM pumped into vacuole & ER by H+ antiport & P-type

Nutrient uptake Ca2+ is expelled by P-type ATPases in PM pumped into vacuole & ER by H+ antiport & P-type enters cytosol via gated channels Nutrient uptake PO43-, SO42-, Cl- & NO3enter by H+ symport

Nutrient uptake PO43-, SO42-, Cl- & NO3- enter by H+ symport also have anion transporters of ABC type Nutrient uptake PO43-, SO42-, Cl- & NO3- enter by H+ symport also have anion transporters of ABC type

and anion channels Nutrient uptake PO43-, SO42-, Cl- & NO3- enter by H+ symport also have anion transporters of ABC type and anion channels Plants take up N many ways

Nutrient uptake Plants take up N many ways: NO3- & NH4+ are main forms Nutrient uptake Plants take up N many other ways NO3- also by channels NH3 by diffusion

NH4+ by carriers Nutrient uptake Plants take up N many other ways NO3- by channels NH3 by diffusion NH4+ by carriers NH4+ by channels

Nutrient uptake Plants take up N many other ways 3 families of H+ symporters take up amino acids Nutrient uptake Plants take up N many other ways 3 families of H+ symporters take up amino acids

Also have many peptide transporters some take up di- & tripeptides by H+ symport Nutrient uptake Plants take up N many other ways 3 families of H+ symporters take up amino acids Also have many peptide transporters some take up di- & tripeptides by H+ symport

others take up tetra- & penta-peptides by H+ symport Nutrient uptake Plants take up N many other ways 3 families of H+ symporters take up amino acids Also have many peptide transporters some take up di- & tripeptides by H+ symport

others take up tetra- & penta-peptides by H+ symport Also have ABC transporters that import peptides Nutrient uptake Plants take up N many other ways 3 families of H+ symporters take up amino acids

Also have many peptide transporters some take up di- & tripeptides by H+ symport others take up tetra- & penta-peptides by H+ symport Also have ABC transporters that import peptides N is vital! NO3- & NH4+ are main forms

Nutrient uptake Metals are taken up by ZIP proteins & by ABC transporters same protein may import Fe, Zn & Mn! Nutrient uptake Much is coupled to

pH gradient Nutrient transport in roots Move from soil to endodermis in apoplast Nutrient transport in roots Move from soil to endodermis in apoplast Move from endodermis to xylem in symplast

Nutrient transport in roots Move from endodermis to xylem in symplast Transported into xylem by H+ antiporters Nutrient transport in roots Move from endodermis to xylem in symplast Transported into xylem by H+ antiporters, channels

Nutrient transport in roots Transported into xylem by H+ antiporters, channels,pumps Nutrient transport in roots Transported into xylem by H+ antiporters, channels,pumps Lowers xylem water potential -> root pressure

Water Transport Passes water & nutrients to xylem s of xylem makes root pressure Causes guttation: pumping water into shoot Transport to shoot Nutrients move up

plant in xylem sap Nutrient transport in leaves Xylem sap moves through apoplast Leaf cells take up what they want

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