Transformation of E. coli
approx. ?/? cells take up plasmid DNA
1 cell/million cells
efficiency of transformation
very inefficient
Competent
when a cell is able to take up a genetically engineered plasmid, ex. E. coli
if a bacterium carries a plasmid without an antibiotic resistant gene
will not be able to grow on that antibiotic
easy to isolate plasmid DNA?
yes
if a single bacterial cell takes up a plasmid molecule
all its descendants will inherit that plasmid too *their genetic makeup will include the genes encoded by the plasmid DNA
restriction enzymes
allow new genes to be added to purified DNA by cutting plasmid at specific nucleotide sides
selective marker
antibiotics can be used as this, to distinguish bateria with and without the plasmid with an antibiotic resistant gene
DNA ligases
attach any foreign genes to the plasmid
scientists use plasmids as
convenient vehicles for introducing new genes into cells
each plasmid contained in a bacterial cell is
duplicated along with the regular chromosome
steps for Competence
1. E. coli cells are exposed to high concentrations of plasmid DNA 2. shocked (briefly exposed to high and then cold temperatures)
a gene that codes for a fluorescent protein can be a
marker for a plasmid
Plasmids
circular pieces of DNA
Selective marker genes allow us to do what?
genes allow us to select between bacteria that have taken up the plasmids from those that have not
Selective Marker Genes
genes that produce an easily detected phenotype ex. resistance to antibiotic, color change in colonies
vector
genetically engineered plasma acts like this delivers new genes to bacterial cells- "delivery system"
where do plasmids naturally occur?
in the cytoplasm of bacterial cells
transformation
introduction of newly recombined plasmid to host cels
what two things are needed to recombine plasmid?
restriction enzymes & ligases
why do plasmids duplicate like this?
so that the plasmid DNA molecules are inherited by both daughter cells each time the parent cell divides
shock allows
some of the cells to take up plasmid DNA and express genes encoded on the plasma THUS CELLS MAY BECOME TRANSFORMED
if a bacterium carries a plasmid with an antibiotic resistant gene
that bacterium will be able to grow and reproduce in the presence of that antibiotic