Transcript Transformation - Workforce3One

Methods for DNA Transfer
Transferring Genes
• Vectors are used to move
genes around
• Plasmids, Bacteriophage,
Cosmids, YACs, BACs,
Viruses are used
• E. coli often used to
express genes that have
been transferred
• Transformation is a
common method for gene
transfer
Transformation
• Recipient cells take up foreign DNA from
surrounding media
• Often accomplished using plasmid vectors
• Artificially induced in laboratories
• Allows introduction of unrelated genes into
bacterial expression systems
• Products of interest can be produced,
extracted, and purified for use
Bacterial Plasmids
• Circular pieces of
DNA that can be
replicated outside the
bacterial chromosome
• Occur in varying sizes
• Capable of carrying
varying sizes and
types of genes
• May produce several
hundred copies in a
single cell
Vector Creation
• Restriction enzymes are used to cut DNA
to be inserted into small fragments
• Plasmids are cut open with REs so DNA
fragments may be inserted
• Plasmids, DNA fragments, and DNA ligase
are mixed to put it all back together:
cloning
• Ready for transformation now
Restriction enzyme
recognition sequence
G A AT T C
C T TAAG
DNA
Restriction enzyme
cuts the DNA into
fragments
G
C
G
Sticky end
Addition of a DNA
fragment from
another source
C
G
Two (or more)
fragments stick
together by
base-pairing
G A ATT C
C T T AA G
G A AT T C
C T T AA G
DNA ligase
pastes the strand
Recombinant DNA molecule
G
C
Vector Creation
Transformation
• Transformation was
discovered in 1928
by Frederick Griffith
using S. pneumoniae
in mice:
“Transforming
Principle”
• In 1944 a genetic basis
was for process was
discovered by Avery,
McLeod, and McCarty
– They named the process
“Transformation”
Transformation of E. coli
• Cells capable of taking up foreign DNA are
competent
– Some cells are naturally competent, some cells have
to be made competent
– E. coli not naturally competent
• Artificial competence induced using cold and
cationic solutions (cold CaCl2)
• Plasmid introduced into iced solution
• Cells heat shocked to force plasmid uptake
• Solution allowed to return to ambient
temperature, pores close
Human cell
E. coli
Isolate DNA
from two sources
Cut both DNAs
with the same
restriction enzyme
Plasmid
DNA
Gene V
Sticky ends
Mix the DNAs;
they join by
base-pairing
Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
Put plasmid into bacterium
by transformation
Recombinant
bacterium
Clone the bacterium
Bacterial clone carrying many
copies of the human gene
Detecting Transformation
• Many plasmids carry antibiotic
resistance genes: R factors
• Used to select transformants
• pBestLuc plasmid contains
ampr and luc genes
• Ampicillin resistance,
Luciferase production
• Potential transformants plated
on ampicillin-containing media
• Colonies exposed to luciferin
solution and observed for
luminescence
Manipulating Genes for
Biotechnology
•
•
•
•
To make products
To improve crops
To improve livestock
To improve quality of
life
• To treat disease
Biotechnology Applications
• Recombinant pharmaceutical products
• Transgenic animals as pharmaceutical
“factories” and organ sources
• Transgenic crops
Bacterium
Cell containing gene
of interest
Plasmid
isolated
DNA
isolated
Gene inserted
into plasmid
Bacterial
Plasmid
chromosome
Recombinant DNA
(plasmid)
DNA
Gene of
interest
Plasmid put into
bacterial cell
Recombinant
bacterium
Cell multiplies with
gene of interest
Copies of protein
Copies of gene
Clone of cells
Gene for pest
resistance
inserted into
plants
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to
make snow
form at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy