Week 12

• Biotechnology (tech that uses biological systems) has a number of applications
• Genetic testing


  • Determine genetic risk factors and ancestry
• DNA profiling


  • Identify DNA at crime scene investigations
• Recombinant DNA, Gene Therapy, Genetic Engineering


  • Express genes from one organism in another

  
• Biotechnology


  The use and application of biological processes such as DNA replication; especially microbial systems for industrial or other purposes
• Biotechnology involves DNA and RNA manipulation
• Scientists are able to isolate, identify and sequence DNA using the principles we have explored in this class
• DNA and RNA have similar physical structure and chemistry when comparing organisms
• Genetically engineered organisms


  Transgenic organisms with recombinant DNA
• Transgenic or genetically modified organisms (GMOs)


  • Sheep that produce a human protein in their milk used to treat emphysema
  • Chickens that produce eggs containing human antibodies to help fight bacterial pathogens
  • Crops like corn, canola, and cotton that have been engineered to resist insect pests
• Recombinant DNA is made by ligating DNA fragments from different organisms


  1. Requires a fragment of double-stranded DNA that serves as the donor

  2. For protein coding genes, reverse transcriptase is used to make double-stranded complementary DNA (cDNA) from mRNA

  3. Requires a vector (carrier) sequence into which the donor fragment will be inserted

  4. Involves cutting DNA (both donor and vector) with the same restriction enzyme so that the donor DNA and the vector DNA have complementary ends for renaturation and ligation (DNA ligase)

  5. The recombinant plasmid (DNA) is transformed into bacteria and replicated

  
• Polymerase Chain Reaction (PCR)


  Amplifies specific DNA sequences

  
• Each cycle of PCR amplification


  1. Denaturation

  2. Annealing

  3. Extension
• Taq polymerase (used in PCR) is a DNA polymerase that is thermostable and named after the bacterium (Thermus aquaticus) it was isolated from
• Enzymes


  • They are able to reduce the activation energy by stabilizing the transition state
  • The rate of the reaction increases because the activation energy is reduced
• Enzyme active site


  • Binds the substrate (S) and converts the substrate to the product (P)
  • The interactions between the substrate and the active site are weak noncovalent interactions or transient covalent bonds
• How enzymes increase reaction rates


  • Physically aligning reactants in the orientation needed for the reaction to proceed
  • Stabilizing the transition states of reactants through temporary chemical interactions
• To get from reactants to products


  1. Reacting atoms have to go through a transition state

  2. Transition state has a higher free energy than the reactants

  3. This is called the activation energy (EA)
• Activation energy


  When the activation energy is low, the reaction is faster
• Enzymes


  • Able to reduce the activation energy by stabilizing the transition state
  • The rate of the reaction increases because the activation energy is reduced
• Enzyme binding substrate(s) at the active site


  1. Enzyme active site binds the substrate (S)

  2. Enzyme converts the substrate to the product (P)

  3. Interactions between substrate and active site are weak noncovalent interactions or transient covalent bonds
• How enzymes increase reaction rates


  • Physically aligning reactants in the orientation needed for the reaction to proceed
  • Stabilizing the transition states of reactants through temporary chemical interactions
• Enzyme active site


  • Extremely small compared to the enzyme itself
  • Specific for both the substrate and the type of reaction that is catalyzed
• Enzymes catalyze specific chemical reactions at the rate the cell needs to survive
• The protein is unchanged by the chemical reaction it catalyzes
• Enzymes are generally proteins but can be nucleic acids
• Enzymes have optimal pH, temperature, and ionic strength
• Enzymes can be regulated
• Enzyme modifications


  • Reversible e.g. phosphorylation
  • Irreversible e.g. covalent bond cleavage
• Enzyme activators


  • Turn on enzymes when their products are needed and the reactants are present
  • Can be allosteric
• Enzyme inhibitors


  • Turn off enzymes when their products are not needed
  • Decrease enzymatic activity
  • Reversible - Form weak bonds with the enzyme
  • Irreversible - Form covalent bonds with the enzyme
  • Can be competitive or allosteric
• Allosteric regulators


  • Bind outside of the active site
  • Usually structured differently from the substrate
  • Can be activators or inhibitors
• Competitive inhibitors


  • Usually structured similarly to the substrate
  • Can be overcome by adding more substrate
• Enzymes that are regulated by allosteric regulators are called allosteric enzymes
• Enzyme inhibitors used clinically


  • To treat many disorders
• Isoleucine binding to the first enzyme in the pathway


  Shuts down isoleucine production
• Type of regulation described

  Allosteric inhibition
• The activation energy (Ea) of the uncatalyzed reaction is higher than the activation energy (Ea) of the enzyme-catalyzed reaction
• The ΔG for the uncatalyzed hydrolysis of sucrose is -29 kJ/mol
• The ΔG for the hydrolysis of sucrose when it is catalyzed by sucrase is the same as the uncatalyzed reaction