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