Transcript Genetic Engineering

Exam 2 T 10/30 at 7:30-9pm
Review Th 10/25 at 5-7pm in WRW 102 and/or
in class
Bonus #1 due 10/25 in class
Genetic Engineering: Direct manipulation of DNA
Bacteria can be modified or serve as intermediates
a typical bacteria
Bacterial DNA
plasmid DNA
A typical
bacterial plasmid
used for genetic
engineering
Moving a gene into bacteria via a plasmid
What problems exist for expressing eukaryotic
gene in bacteria?
Bacterial DNA
plasmid DNA
Reverse
transcriptase
can be used to
obtain coding
regions without
introns.
After RT, PCR will
amplify the gene or DNA
Fig 20.14
Moving a gene into bacteria via a plasmid
RT and PCR
Restriction Enzymes cut DNA at specific sequences
Restriction enzymes cut DNA at a specific
sequence
Cutting the
plasmid and insert
with the same
restriction enzyme
makes matching
sticky ends
Fig 20.2
A typical
bacterial plasmid
used for genetic
engineering
Using sticky ends to add DNA to a bacterial plasmid
Fig 20.2
If the same
restriction enzyme
is used for both
sides, the plasmid
is likely to religate
to itself.
Fig 20.2
The plasmid is
treated with
phosphatase to
remove the 5’-P,
preventing selfligation
Fig 20.2
Transformation of bacteria can happen via
several different methods.
Fig 20.8
Bacteria can take up DNA from the environment
Fig 7.2
Transformation of bacteria can happen via
several different methods all involving
perturbing the bacterial membrane:
•Electroporation
•Heat shock
•Osmotic Stress
Fig 20.8
How can you know which bacteria have been
transformed, and whether they have the insert?
Fig 20.5
Resistance genes allow
Figurebacteria
20-5 with the
plasmid to be selected.
Bacteria with the resistance
gene will survive when
grown in the presence of
antibiotic
Fig 20.5
Is the insert present?
FigurePlasmids
20-5 with the MCS
in the lacZ gene can be
used for blue/white
screening…
A typical
bacterial plasmid
used for genetic
engineering
Fig 20.5
Intact lacZ makes a
Figureblue
20-5
color when
expressed and provided
X-galactose
Fig 20.5
When the lacZ gene is
Figuredisrupted,
20-5 the bacteria
appear white
Blue/white
screening:
Transformed
bacteria plated on
antibiotic and Xgal plates.
Each colony
represents millions
of clones of one
transformed cell.
Successful transformation
will grow a colony of
genetically modified
bacteria
Fig 20.4
RT and/or
PCR
Inserting a gene into a
bacterial plasmid
Millions of Hectares
Bacteria can be used to transform plants
Global area planted
with GM crops
http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html
Texas =
70 ha
Agrobacterium infect plants, inserting their
plasmid DNA into the plants genome.
Agrobacterium infect plants, inserting their
plasmid DNA into the plants genome.
Fig 20.24
By replacing the gall forming genes with other
DNA when the Agrobacterium infect a plant, it
will insert that DNA into the plant.
Fig 20.25
The generation of a transgenic plant
Grown on herbicide
Fig 20.26
How do you know whether the gene you want
to express has the correct sequence?
How do you know whether the gene you want
to express has the correct sequence?
DNA sequencing
• The structure of 2’,3’-dideoxynucleotides
Fig 20.15
The dideoxy
sequencing method
Fig
20.16
The dideoxy
sequencing
method
Fig
20.16
Gel produced by the
dideoxy sequencing
method
Fig
20.16
Computerized sequencers use a similar method
Fig
20.17
• Creation of Caenorhabditis elegans transgenes
Figure 20-28
Creation of
Drosophila
melanogaster
transgenes
using a
transposon
Fig 20.29
• Creation of Mus musculus transgenes
Figure 20-30
Fig 20.30
…now enjoy
making
Frakencritters.
Exam 2 T 10/30 at 7:30-9pm
Review Th 10/25 at 5-7pm in WRW 102 and/or
in class
Bonus #1 due 10/25 in class