Lesson 10 Biotechnology Flashcards
Experiment 19A: Human Peptide in Bacteria
Hypothesis
It is possible to produce human somatostatin in a recombinant bacterium.
Experiment 19A: Human Peptide in Bacteria
Starting materials
Normal E. coli strain that was unable to synthesize somatostatin, and bacterial plasmids that carry the ampR gene along with bacterial promoters.
Experiment 19A: Human Peptide in Bacteria
protocol
- Synthesize 8 oligonucleotide that will hybridized to each other when mixed together
- Use DNA ligase to covalently linked the oligonucleotide so they form a molecule
- Insert fragment into a plasmid by digesting the plasmid at unique sites for EcoRI and BamHI
- Transform plasmids into E. coli. Spread on plates with ampicillin and grow overnight.
- Grow the recombinant bacteria and activate the lac promoter with isopropyl thiodigalactoside.
- Place in tub and centrifuge to obtain pellet
- Resuspend pellet in 70% Formica acid and cyanogen bromide. This breaks open cells and cleaves polypeptides as methionine residues.
- Use radioimmunoassay to determine amount of somatostatin.
Experiment 19A: Human Peptide in Bacteria
Interpreting the data
Recombinant bacteria carrying somatostatin gene produced somatostatin.
Biological control
Involves the use of microorganisms or products from microorganisms to alleviate plant diseases or damage from undesirable environmental conditions
bioremediation
Use of microorganisms to decrease pollutants in the environment
biodegradation
The backbone structure of a toxic pollutant is destroyed to yield metabolites that are less complex and nontoxic
biotransformation
Rendering something less toxic without degradation, but with oxidation or reduction reactions
Two characteristics of stem cells
- Can divide
2. Can differentiate into one or more different cell types
Gene redundancy
When one type of gene is inactive, another gene with a similar function may compensate for the inactive gene
Molecular pharming
Strategy for getting transgenic livestock
How to express human gene in cow’s milk?
clone the gene next to a milk-specific promoter. Protein encoded by human gene is expressed within mammary gland and secreted in milk. Then can be isolated from milk.
What makes FlavrSavr tomatoes last longer?
They express antigens RNA, which silences the expression of the gene that is necessary for overripening
DNA fingerprinting
A technology to identify a particular individual based on the properties of their DNA. Can determine genetic relation.
How is DNA separated for fingerprinting?
Gel electrophoresis
minisatellites
Locations within the chromosomes that contain randomly repeated sequences. Where DNA fragments are found.
VTNR
Loci with variable number of tandem repeats
microsatellites
Variably repeated sequences in genome; smaller than VNTRs an can be easily amplified by PCR.
How can microsatellites amplified by PCR be detected?
With Fluorescently labeled PCR primers
Uses of DNA fingerprinting
method of identification and relationship testing
Gene therapy
Introduction of cloned genes into living cells in an attempt to cure disease
Two ways to introduce DNA into cells
Liposomes or viral vectors
liposomes
Lipid vehicles that are naturally taken up by cells via endocytosis
Adenosine deaminase
enzyme that is involved with purine metabolism. If both copies of the ADA gene are defective, it leads to a disease called severe combined immunodeficiency disease (SCID)
First disease to be treated by gene therapy
Adenosine deaminase deficiency
How to get gene to lung cell
- Aerosol spray
2. Liposome delivery
Delivery by aerosol spray
Normal CF gene is cloned into an adenovirus, which normally infects lung epithelial cells and cause infection.
Liposome delivery of CF gene
Normal CF gene is contained within liposomes. When inhaled by patient, they are taken up by the lung epithelial cells.
