Devise a restriction analysis method to confirm the desired recombinant; use a single most appropriate enzyme. Calculate the expected sizes of the restriction fragments from each, the vector and the desired recombinant

Know all of the hormones that have their effects on the kidneys, and what those specific effects are, and know about the hormones and enzyme (Renin) released by the kidneys and their target tissues and effects at the various tissues.

1. Please explain why “noncompetitive inhibition”- where it is observed experimentally- is more likely to be a coincidence than a reveal of something profound in the mechanism of an enzyme (in contrast to competitive and uncompetitive inhibition).

Ocean pout, a fish found in cold waters, produce an antifreeze protein that allows them to survive at near freezing temperatures. Your company plans to genetically engineer bacteria to grow antifreeze proteins to improve the storage of ice cream products

a) The vector and PCR product that you will use to clone the antifreeze gene are shown above along with the locations of three different restriction enzyme cut sites Which restriction enzyme (Hindill, BamH1, or EcoR1) will allow you to successfully clone this transgeno? Brietly justify your answer.

b) Describe how you will select for bacteria that carry the recombinant DNA molecule once you have completed the transformation

Experiment 3: Cloning a DNA Fragment into a Bacterially-Derived Plasmid Vector

Foregin DNA = 720 Plasmid DNA 2804

Post-Lab Questions

1. What is the expected size of the plasmid plus the cut foreign DNA?

2524 base pairs

2. What type of ends do the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning?

3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does this enzyme work?

4. How would you clone a gene into a plasmid if there were no common restriction sites between the two DNA sequences?

DIPF and Sarin and "organophosphate" insecticides are all irreversible inhibitors of enzymes with a "special" -- highly reactive -- active-site Ser residue. Which of the following is true about DIPF (or Sarin or organophosphate insecticides)?

1. Because plant and animal life on earth is impossible without photosynthesis

A. environmental pollution is not a problem

B. there is concern for increasing numbers of plant species that face extinction

C. worldwide decline of insect diversity is of no concern

D. there is no need to consider conservation biology

2. Reactions with a positive ΔG

3. The induced fit is an improved model for enzyme function than the lock and key model because it considers

A substrate is decomposed in the presence of an enzyme according to the Michaelis-Menten equation
with the following kinetic parameters:
Km = 20 g/L
Vmax = 12.0 g/L-min
(a) Determine the concentration of substrate after leaving the second reactor in a two-reactor series of
20-liter CSTRs. The flow rate is 2.00 L/min. The inlet substrate concentration is 40 g/L. The
enzyme concentration in the two reactors is maintained at the same value all of the time.
(b) Determine the conversion in terms of substrate conversion of the two-reactor system and the
conversion of a single CSTR equal to the total volume of the two-reactor system. Clearly indicate
the configuration with the greater conversion

Exercise : Enzyme-Linked Immunosorbent Assay (ELISA)

At the conclusion of this exercise you should be able to:

• explain the principles of the enzyme-linked immunosorbent assay (ELISA).
• determine the qualitative results of a clinical scenario using ELISA.
• understand the purpose of the reagents used in ELISA.

Background

Immunology is the study of the immune response. This involves the interaction between antigens, antibodies, and cells. Immune responses are generated by the body to recognize substances considered to be “foreign”. Microorganisms can generate different immune responses when they infect a host. This leads to the formation of antibody in a host. This exercise will explore some of the basic principles of immunology using an indirect ELISA to identify the presence of antigen in a sample of serum.

Scenario

A serum sample was obtained from a patient who was affected by a severe gastrointestinal infection, which was presumed to be caused by a bacterial infection. In order to investigate the causative agent, an ELISA using antibodies of known intestinal pathogens was performed to identify the microorganism that caused this infection.

Materials

1. A sample of serum was obtained from the child.
2. Antigens obtained from three known gastrointestinal pathogens:

Shigella sonnei, Escherichia coli, and Salmonella typhimurium

3. Microtitre polystyrene plate.
4. Coating buffer (phosphate buffer solution, pH 9.6; PBS).
5. Blocking buffer (PBS/Tween)-Tween is a detergent added to buffer to prevent non-specific binding of molecules.
6. Secondary antibody (anti-human immunoglobulin). This antibody is attached to an enzyme that will react with a substrate that is added in the last step of the process.
7. Enzyme substrate: this is used in immunoassays. For this assay, the substrate used is horseradish peroxidase, which causes a yellow reaction once it attaches to the secondary antibody.

Procedure

1. Add 50 µl of each of the three antigens diluted in coating buffer to rows A-C and columns 1-12 of a microtiter plate.

Row A: Shigella sonnei

Row B: Escherichia coli

Row C: Salmonella typhimurium

2. After incubating for 1 hour at room temperature remove liquid by tapping onto paper towel.
3. Add 300 µl blocking solution to each well and leave for 5minutes.
4. Tap out residual solution.
5. Add 50 µl of PBS/Tween to rows A-C and columns 1-12.
6. Add 50 µl of the patient’s serum to column 2 and rows 1-3.
7. Proceed to make two-fold dilutions in each row by removing 50 µl from column 2 and transferring to column 3. Continue until the last column is reached (column 12).
8. Incubate the plate at room temperature for 1 hour.
9. Empty plate by tapping out fluid on a paper towel.
10. Fill each well with wash solution (PBS/Tween), invert and remove residual liquid. Repeat 3 times.
11. Add 50 µl of secondary antibody to each well and incubate 1 hour at room temperature.
12. Remove residual liquid as described above and complete 3 washes as above.
13. Add 50 µl of substrate to each well and note the change in colour in each well.

Results

The results of this experiment are shown below.

Questions

1. Which organisms was the causative agent of infection for this patient? Explain you answer in comparison to the other samples provided.

2. What is the purpose of using a negative control? Explain what it means of column 1 wells appear yellow.

3. What are the fundamental differences between direct and indirect ELISA methods?

4. Explain the purpose of diluting the serum sample provided.

5. Describe the purpose of each of the components in the ELISA.