MI EOC 4.1

What role does insulin play in diabetes?

- Insulin allows cells to take up sugar, getting sugar out of the blood
- Type 1 diabetics need insulin injections to survive—otherwise their cells will die (need glucose to make energy)

How has the diagnosis and treatment for diabetes changed in the last 200 years?

- Long ago: diagnosed by tasting pee for sweetness
- 1700's-1800's: treated with dietary restrictions & deadly chemicals
- 1920s on: insulin used to treat patients

How can bacterial plasmids be used to produce proteins such as insulin?

- This is done through genetic engineering
- Plasmids are isolated from bacteria
- Plasmid is cut with restriction enzyme
- The gene for insulin (or other protein) is inserted into the plasmid
- Plasmid is closed (it now contains recombinant DNA—DNA from multiple sources)
- Bacteria are made to take up plasmid and pump out protein

What is bacterial transformation?

Transformation is the process by which cells are made to take up a genetically engineered plasmid.
- after that they'll make the protein that the inserted gene(s) code for
- Transformed bacteria are grown on antibiotic like ampicillin to ensure purity of culture

How can you gauge the success of a transformation experiment?

You have to calculate the transformation efficiency, which how many transformants there were per microgram of DNA added

How does amino acid structure relate to the overall shape of a protein?

- Proteins are made of amino acids, some of which are hydrophilic (water-loving) and some of which are hydrophobic (water-fearing)
- In an aqueous environment proteins fold so that the hydrophobic amino acids "hide" on the inside
- In a hydrophobic environment, proteins fold so that the hydrophobic amino acids coat the outside

What is chromatography?

A way of separating the parts of a mixture, based on their different properties.

How can chromatography be used to separate proteins?

Proteins can be passed through column chromatography, with some columns grabbing more hydrophilic proteins and others grabbing more hydrophobic proteins.

How can electrophoresis be used to check the purity of a protein sample?

- The protein is loaded into a well and standards are in another well
- The protein travels and so do the standards
- The protein can be compared to the standards and its length determined
- Protein length is compared to known length of target protein (i.e. insulin)

What is SDS-PAGE?

- SDS stands for Sodium Dodecyl Sulfate & is a soap that dissolves hydrophobic molecules (like GFP)
- PAGE stands for PolyAcrylamide Gel Electrophoresis is the method used to check for protein purity

How does protein electrophoresis differ from DNA electrophoresis?

DNA electrophoresis:
- Uses agarose gel
- Requires PCR of DNA first
- Gel is loaded horizontally
- DNA moves across the gel

Protein electrophoresis:
- Uses polyacrylamide gel
- Requires SDS application to protein 1st (at least in case of GFP)
- Gel is loaded vertically
- Protein moves down the gel

What biomedical professionals are involved in all stages of producing and manufacturing a protein product?

- Genetic engineers isolate & insert the gene
- Microbiologists culture the bacteria
- Lab technicians generate/purify the protein
- Pharmacologists design/test the medications

How does a cover letter differ from a resume?

Cover Letter:
- Emphasizes particular info from resume
- Complete sentences
- Details about a few things you've done or skills you have
- More targeted to individual employer

Resume:
- More info with less details than cover letter
- Bullets, phrases
- A snapshot of who you are and what you've done
- More "one size fits all"