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Isobutanol Production (Over expression of Isobutanol Production Pathway…
Isobutanol Production
Over expression of Isobutanol Production Pathway
Ethanol Production (fermentation)
pyruvate -> acetaldehyde -> ethanol
Key Enzymes
Acetolactate synthase
acetohydroxy acid isomeroreductase
dihydroxy-acid dehydratase
2-ketoacid decarboxylase
alcohol dehydrogenase
Quantification
Bradford
at low pH, proteins pulls red and green into blue form and stabilize blue
Activity Assay
carry our reaction utilizing ADH, measure absorbance of NADH
colorimetric enzyme-based coupled assay
1st
isobutaol -> isobutyraldehyde; coupled with NAD+ -> NADH
NADH is based on
2nd
1-methyl PMS -> reduced form; coupled with NADH + NAD+
pass on electron
3rd
MTT -> MTT formazan (purple)
MTT formazan accumulates to an amount that reflects isobutanol present in the beginning
Over expression mechanism
Bacteria growth curve
induce at log phase as it has most ribosome and most active
Plasmid with lac operon
Production
isobutanol product pathway
split required enzyme into 2 plasmids
different Origin of replication
both are replicated at the same rate
different section marker
both plasmids are obtained
control: normal cellular isobutanol production
Minimal M9 medium
carbon
glucose
backbone for everything
phosphorus
Na2HPO4, K2HPO4
ATP and phospholipids
Nitrogen
NH4Cl
N compounds
Sulphate
FeSO4, MgSO4
enzyme cofactors, amino acid
Ion
Co, Ca, Zn, CaCl2
vitamin, signaling
Amp or Kan
check expression
inoculate
induce and grow
measure OD600 (normalize later)
get supernatant
Organisms for biofuel production
Native Biofuel Producers
Advantages
existed fuel production pathway
Challenges
not easily genetically modified
simple sugar only
low tolerance to fuel molecules
Cellulolytic Organisms and Thermophiles
Challenges
not easily genetically modified
need to introduce production pathway
Advantages
can degrade biomass
high temperature->good for biomass degradation and biofuel isolation
Model Organisms
Challenges
can't digest biomass, simple sugar only
need to introduce 2 pathways
Advantages
known metabolic networks
high tolerance to fuels
easy gene tool
Autotrophic Organisms
Advantages
CO2 required as only carbon source
Challenges
not easily genetically modified
introduce proaction pathway
scale-up
ABE Pathway
acidogenesis
produce acetate and butyrate
EXPONENTIAL GROWTH
solventogenisis
use acetate and butyrate to produce ethanol and butanol
STATIONARY PHASE
glycolysis
produce pyruvate and other following products
ALWAYS ON
Higher order alcohols
upreguate amino acid biosynthesis
kept acid decarboxylase
alcohol dehydrogenase
Use Proteins
free the carbon backbone
channel them to alcohol production
Take ammonia off
mutate enzymes in transamination
prevent amino going back to carbon backbone
Use Fatty Acid
beta-oxidation in reverse
Key enzymes
AcetylCoA carboxylase
make FA precursor
OVEREXPRESS
acyl-ACP feedback inhibits
Thiolase
make FA from acyl-ACP
OVEREXPRESS
so no acyl-ACP accumulation that feedback inhibits acylCoA carboxylase
enzymes in beta-oxidation
MUTATE
synthesized FA won't be degraded