MI unit 4

4.1.1: What were the steps of learning about diabetes?

Went from “better diet” to testing blood sugar levels with a tablet in your urine, then a test strip to dip in pee to see if it changed color (high sugar), to a “new invention called insulin” which they could inject to help lower blood sugar levels, then pills to stimulate pancreas to release insulin, then a small glucose meter to actually measure the real amount, then a pump that keeps insulin steady, and now apps on a phone that can monitor it.

4.1.2: How does a chemical transformation work?

This means that we are inserting a plasmid into a bacteria. For the lab, we inserted GFP into a plasmid and performed a heat shock. The heat shock causes bacteria/cells to expand and become more permeable, which allowed the plasmid with GFP to enter E.coli bacteria. We now put the bacteria we had onto a plate with ampicillin. Only the bacteria with the plasmid will grow on the plate since it also contains a gene (Amp) that is ampicillin resistant. The plasmid also contains IPTG which allows the GFP to work and glow fluorescent green under UV light, helping us know that the gene was expressed correctly in the transformed bacteria.

4.1.2: explanation of the PflouroGreen Plasmid Map

GFP: lets bacterial cells produce the flourescent green light

T7 promotor: contains IPTG which allows an RNA polymerase to access the promotor and “turn on” the GFP which lets it glow

Ori: DNA sequence that allows bacteria to initiate plasmid copying

Amp: allows the transformed bacteria to be ampicillin resistant

Lab explanation Module 1:

1. Add plasmid with GFP and Amp to E.coli bacteria

2. make bacteria competent with calcium chloride (CaCl): Makes the bacterial membrane more permeable to help plasmid DNA stick to the cell surface of the bacteria, think “loosens the membrane so DNA can get in”

3. Heat shock: creates a sudden pressure difference which makes the membrane expand and more permeable to allow the plasmid DNA in the bacteria cell

4. Recovery broth: Gives bacteria time to repair and start expressing genes

5. Ampicillin Plate: Only bacteria with the plasmid will survive (remember, it contains Amp, the ampicillin resistant gene)

6. IPTG plates: the plates containing IPTG turn on the T7-promotors in the plasmid which allows the GFP in the plasmid to turn fluorescent green

Lab Module 2 explanation:

Purpose: Break open the bacteria cells so we can isolate the GFP by getting it out of the cells

1. Use lysis to break open bacteria cells

2. Centrifuge and we want Supernatent (has GFP protein)

Lab module 3 explanation:

Purpose: purify GFP protein

1. Use column chromatography (separates GFP from other proteins)

   1. Proteins move at different speeds because they react differently to the beads which allows all the GFP to be in one place and become isolated from the other proteins

Types of kidney Interventions:

1. Hemodialysis: interferes with life because you have to go in to the doctors a lot, placed on the arm to filter toxins from the blood (pulls blood, filters, injects)

2. Peritoneal Dialysis: can do from home, soft flexible catheter placed in abdominal cavity, filters waste by draining fluid and replacing it

3. Kidney transplant

Amino Acid structure

1. Hydrophobic (non-polar): stays on the inside to avoid water

2. Hydrophilic (polar): stay on the outside

3. Acid(-) base(+) pairs: attract to each other because of opposite charges

4. Cysteine group: form a covalent bond with each other

Tissue-Typing

• All humans have HLA

  • can stimulate immune responses

• HLA typing identifies which antigens are present in recipient and donor

  • closer match means immune system is less likely to reject the organ

• Set of HLA antigens received from a parent in called a haplotype

• during testing, 6 HLA antigens are tested for each person, and a 6-antigen match is the best result

Antibody screening (PRA) and crossmatching

• PRA: mix recipients serum with 60 others to see how many HLA antibodies patient has

  • Less is better

• Crossmatching: mis donors white blood cells with recipients serum to see if recipient has any antibodies to donors HLA

  • No reaction means kidney will likely not be rejected

What molecular weight did we want the GFP to be about?

20,000 to 30,000

3 types of immunosuppressants:

1. calcineurin inhibitors: inhibits calcineurim, a protein that activates T-cells which attack foreign antigens

2. Antiproliferative: inhibits growth of T-cells

3. corticosteroids: ant-inflammatory agents that reduces overall activity of immune system