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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
isolate a vector
DNA is cleaved by an enzyme
Gene of interest is inserted into the vector
Vector is taken up by a cell (ex. bacterium)
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
