Thursday, October 20, 2011

1.3 An Intro to Metabolism & 1.4 Enzymes

energy = ability to do work
catabolic reactions: breakdown of complex substances (ex)
anabolic reactions: synthesis of complex substances from smaller units (end)
metabolism = catabolic + anabolic
Potential energy is stored energy; Kinetic energy is used
work = transfer of energy
First Law of Thermodynamics: Energy can neither by created or destroyed, but converted
Energy is absorbed when bonds break
Energy is released when bonds are formed
entropy = measure of randomness/disorder ∆S ∝ disorder
free energy = energy that can do useful work
∆S ∝ 1/free energy
Second Law of Thermodynamics/Law of Entropy: disorder of the universe increases with any change
dead cell = when all its reversible reactions have reached equilibrium (blood/buffer?)
phosphorylation = attaching phosphate group to an organic molecule (synthesis of ADP + Pᵢ --> ATP)
OIL = oxidation is losing
RIG = reduction is gaining

enzymes = protein catalysts
lowers activation energy
substrate = substance catalyzed by an enzyme
active site = location where the substrate binds to
substrate + active site = enzyme-substrate complex
cofactors: Zn²⁺; Mn²⁺
coenzymes: NAD⁺ NADP⁺
competitive inhibitors = similar to substrate; blocks substrates to bind to active site
noncompetitive inhibitors = attaches to another site on enzyme --> changes shape of active sites
allosteric sites = receptor sites to inhibit/promote an enzyme's activity
activator = promotes (stabilizes active form)
allosteric inhibitor = inhibits (stabilizes inactive form)
feedback inhibition = substances catalyzed later on can be used as inhibitors

Saturday, October 15, 2011

Biotech Test

That's right!  Here's another one...

Vector Cloning:

  1. Isolate gene of interest
  2. Cut using restriction enzymes (2 cut sites)
  3. Take plasmid from bacteria
  4. Cut using the SAME restriction enzymes (1 cut site)
  5. Put gene of interest into plasmid using the sticky ends, glued together by ligase
  6. RECOMBINANT DNA/ TRANSFORMATION
  7. Culture
  8. "Infect"
How to isolate the gene of interest: gel electrophoresis
Fragments get cut at various lengths.  Moves from NEGATIVE --> POSITIVE
Complete digestion: all fragments cut at restriction site
Incomplete digestion: all possible fragments are cut

VECTOR CLONING EXPRESSES THE GENE
Agarose Gel Electrophoresis Video

PCR WILL NOT

Polymerase Chain Reaction:
  1. Denature DNA, break hydrogen bonds
  2. DNA primers, dNTPs & Taq polymerase
  3. Repeat
Replication is EXPONENTIAL after the THIRD CYCLE

Restriction Fragment Length Polymorphism
R -restriction enzymes
F- complete digestion
L -PCR
P- patterns (comparison)

DNA Sequencing (Sanger Method)
  1. Denature DNA, break hydrogen bonds
  2. DNA primers, dNTPs, ddNTPs & Taq polymerase
  3. ddNTPs stop replication
  4. PCR
  5. Complementary sequence read from bottom to top