the wild world of biotechnology!!. applications genetic transformation cloning - genes and entire...
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The Wild World of The Wild World of Biotechnology!!Biotechnology!!
ApplicationsApplications
Genetic Transformation
Cloning - Genes and entire organisms
Gene Therapy
Environmental Clean-Up
How do we genetically How do we genetically engineer or modify an engineer or modify an
organism?organism?
Selective breeding
Genetic transformation
Genetic Genetic Transformation Transformation
This is the process by which we get an organism to express foreign DNA
e.g. making a tomato synthesize antifreeze proteins that are commonly found in fish
e.g. making a bacterial cell synthesize human insulin
Recombinant DNARecombinant DNA
Recombinant DNA = a molecule of DNA that contains DNA from two or more organisms
Making recombinant DNA is the first step in producing genetically engineered (GE) or genetically modified (GM) organisms
Making Recombinant Making Recombinant DNADNA
Isolate the gene of interest (e.g. the gene for insulin) using restriction enzymes
This is a hit or miss process that requires a great deal of luck to be successful
Restriction enzymes generally cut at palindromic DNA sequences
Making Recombinant Making Recombinant DNA continued...DNA continued...
Some method is needed to get the gene of interest into the cells of the organism we wish to transform
e.g. plasmid vectors and gold particles or electroportation
Polymerase Chain Polymerase Chain Reaction (PCR)Reaction (PCR)
Another DNA cloning technique
Using plasmid vectorsUsing plasmid vectorsThese are small circular bits of DNA that are separate from the main prokaryote chromosome.
They are easily transformed because of there chemistry
Bacterial cells will absorb foreign plasmids and express their genes. You and I cannot do this!!!
pGLOpGLO
pGLO are plasmids that carry the genes for fluorescent proteins from jellyfish
Getting bacteria to Getting bacteria to express pGLO genesexpress pGLO genes
The trick is getting the recombinant plasmid past the cell membrane and into the cell and then getting the cell to express the genes.
We use chemicals (CaCl2) and heat shock to get recombinant plasmids into the cell.
We include antibiotic resistance genes in the recombinant plasmid so that only the successfully transformed bacteria live.
We make sure the gene of interest is near a known operon and we intentionally turn that operon on (e.g. arabinose, tryptophan, lactose)
Gene cloningGene cloning
Once the bacteria take up the recombinant plasmid they will go through DNA replication and, in effect, clone the gene of interest many times
Studying DNAStudying DNAGel electrophoresis
A method for separating nucleic acids based on size and electrical charge
Gel ElectrophoresisGel ElectrophoresisA DNA sample is cut with restriction enzymes.
The cut up DNA is placed in one end of a gel and electricity is passed through the gel
Because DNA carries a negative charge the electric current is able to carry the DNA through the gel
The smallest pieces of DNA move the furthest distance
RFLPsRFLPs
Restriction fragment length polymorphisms
This term refers to variation in a gene sequence in a population
RFLPs can be used to determine paternity, in forensics, to map genes, and to detect alleles
Draw a restriction map Draw a restriction map of the plasmid...of the plasmid...