BIOL0510: Microbial Biotechnology

What is the biotechnology timeline?

• pre-Pasteur (fermenting drinks/food)

• Pasteur (industrial fermentation and organic acids)

• antibiotics

• post-antibiotics (producing amino acids, enzymes, etc)

• synthetic microbio (vaccines, insulin, biofuels)

What is microbial biotechnology?

the use of microorganisms, cells, or cell components to make a product (ex. foods, vitamins, enzymes)

What is erythropioetin?

a hormone that encourages the production of red blood cells; injections of it can be administered

What is a vector?

a DNA molecule used as a vehicle to transfer genetic material (ex. a plasmid or virus)

What are shuttle vs expression vectors?

shuttle: usually plasmids that can propagate in two or more species

expression: transfer a gene and express the protein product of the gene

How are vectors used?

adding DNA encoding for erythropioetin to the plasmid; introducing the plasmid back to a bacterial cell

What is recombinant DNA?

DNA created by combining genetic material from different sources/species to produce a ‘recombined’ genome

What is a transgenic animal?

one whose genome has been altered by the introduction of a foreign gene from another species

Describe the steps of genetic engineering using recombinant DNA technology

1. isolate a vector

2. DNA is cleaved by an enzyme

3. Gene of interest is inserted into the vector

4. Vector is taken up by a cell (ex. bacterium)

5. Cells with gene of interest are cloned

How do we cut and paste a gene of interest into a plasmid?

restriction endonucleases are DNA cutting enzymes that cut at the ends of your gene of interest

Why do bacteria have restriction endonucleases?

• they are antimicrobial against phages

• they can degrade other bacteria that may infect them with their DNA

(defense strategies)

How do we get a new bacterial cell to take up a plasmid? 2 ways

• transformation: CaCl2 + heat shock OR electroporation

• phage infection: transduction

How do we make sure that the bacteria received the plasmid (selecting for the bacteria that took up the plasmid)?

• antibiotic selection

• marker (GFP)

• sequencing

Describe the assay we use to determine if the bacteria took up the Amp resistance?

ASK FOR EXPLANATION

What do we do to the bacteria with recombinant DNA after checking for selection?

purification

• give them media and let them grow

• survey the liquid; filter the purified protein product into a sample

List some drugs that are made via cloning

insulin, human growth hormone, estrogen, steroids, epinephrine

How did diabetic people get insulin treatment before cloning?

• received insulin injections produced by cow pancreases (14 cow pancreases/1 person/year)

How did people with stunted growth get human growth hormone before cloning?

human cadavers; 80 cadavers were needed/person/year; risk: potential transmittance of human pathogens

List pros to protein cloning

• increased safety of supplemental drugs

• a reliable, affordable, and purified source