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Exam 2 M 10/29 at 7-8:30pm in UTC 2.102A
Review Th 10/25 at 5-7pm in WRW 102
Bonus #1 due now
Last day to drop with a ‘Q’…10/24
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