MicroLecE2Ch9 Flashcards
Biotechnology
Use of microorganisms, cells, or cell components to make a product
Exs: foods, antibiotics, vitamins, enzymes
Recombinant DNA (rDNA) technology
Insertion or modification of genes to produce desired proteins
Vector
Self-replicating DNA used to carry the desired gene to a new cell
Plasmid
Most common type of vector
Small circular piece of DNA
Non-essential
Ex F-factor used in conjugation
Clone
Population of cells arising from one cell, each carries the new gene, all identical
Genetic Modification Procedure
Vector is isolated
DNA is cut by a restriction enzyme into fragments resulting in DNA containing gene of interest
Gene is inserted into plasmid
Plasmid taken up by a cell such as a bacterium which transforms the bacterium
Cells w/gene of interest are cloned
Goal is to either make copies of the gene (pest resistance, cleaning up toxic waste) OR to make protein product of the gene (human growth hormone treats stunted growth)
Selection
Allows for selecting which microbe has the desired product
Mutation
Mutagens cause mutations that might result in a microbe w/a desirable trait
Ex: some vaccines
Site-Directed Mutagenesis
Change a specific DNA code to change a protein
Restriction Enzyme/Restriction Endonuclease
First step of cloning is cutting open the plasmid
Each restriction enzyme cuts a specific sequence of DNA
Discovered in bacteria which naturally produce these enzymes as a defense mechanism
Destroys bacteriophage DNA in bacterial cells, protects its own DNA w/methyl groups on the surface
Every restriction enzyme site is specific and a palindrome (reads same forward and backward)
Polymerase Chain Reaction (PCR)
Can make millions of copies of DNA from a small amount of DNA
Requires target DNA, RNA Primer, DNA Polymerase, nucleotides A/T/C/G, ATP
1) First Cycle:
Melting: incubate target DNA at 90 C to separate the strands by melting the hydrogen bonds
Primer Binding: primers attach to single-stranded DNA during incubation at 60 C
Extension: incubate at 70 C, during this time two copies of target DNA are formed
2) Second Cycle:
Repeats the first cycle
Inserting Foreign DNA Into Cells
5 ways –
1) Electroporation: most common. Electroshock the bacteria allowing it to expand and the pores to open
2) Transformation: uptake of naked DNA. Uses a chemical to open the pores
3) Protoplast Fusion
4) Gene gun
5) Microinjection
Genomic Libraries
Made of pieces of an entire genome stored in plasmids, phages, or yeast cells
Complementary DNA (cDNA)
Eukaryote
Makes DNA copy of the mature mRNA that already has intron removed
Then DNA can be stored and active genes are ready for use
Selecting a Clone
1) Plasmid DNA and foreign DNA both cut w/same restriction enzyme. Plasmid has genes for lacZ and ampicillin resistance
2) Foreign DNA will insert into the lacZ gene. Bacterium receiving the plasmid vector will not produce X-gal if foreign DNA has been inserted into the plasmid
3) Recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant
4) All treated bacteria put on plate containing ampicillin and lacZ
5) Only bacteria that picked up the plasmid will grow in presence of ampicillin. Bacteria that hydrolyze X-gal produce blue colonies. Bacteria that cannot hydrolyze X-gal produce white colonies which means it has the actual gene inserted into the plasmid
Making a Product
1) E. coli: easily grown, genomics are known, need to eliminate endotoxin from products, cells must be lysed to get product
2) S. cerevisiase: easily grown, genomics known, may express eukaryotic genes easily
3) Plant cells and whole plants: easily grown, may express eukaryotic genes easily
4) Mammalian cells: harder to grow, may express eukaryotic genes easily
Therapeutic Applications
1) Human enzymes and other proteins
2) Subunit vaccines
3) Nonpathogenic viruses carrying genes for pathogen’s antigens as DNA vaccines
4) Gene therapy to replace missing or defective genes
RNA Interference (RNAi)
Occurs in cytoplasm
Used to silence a gene causing problems
Interfered w/ability of the mRNA to bind to the ribosome
Random Shotgun Sequencing
Tells the nucleotide sequence but not where gene is located or fxn 1) Constructing a gene library: isolate DNA cut DNA w/restriction enzymes clone DNA in a BAC 2) Random Sequencing: sequence DNA fragments 3) Closure phase: assemble sequences edit sequences, fill in gaps
Human Genome Project
Goal was to discover the nucleotide sequence
DNA Electrophoresis
Agarose gel - square of agar
1) put holes in agar aka “wells” towards the top and put DNA in those wells
2) run electric charge through the gel and the DNA will migrate through the gel towards the positive end/bottom of the gel b/c DNA is negatively charged
3) shorter pieces of DNA will migrate faster and be closer to bottom end of the gel, longer pieces of DNA will migrate slower and be closer to the well
EcoR1
PCR used more now than DNA Electrophoresis b/c faster and more accurate
Southern Blotting
Same as DNA Electrophoresis except at the end the filter is exposed to a radioactively labeled probe for a specific gene. Probe will base-pair w/a short sequence on the gene
Filter is exposed to X-ray film. Fragment containing the gene of interest is ID’d by a band on the developed film
Agrobacterium
How plants are genetically modified. Produces a plant that has the agrobacterium in it
