Genetic Engineering: Direct manipulation of DNA Bacteria can be ...

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Dec 12, 2012 (4 years and 4 months ago)

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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

Bacterial DNA

plasmid DNA

What problems exist for expressing eukaryotic
gene in bacteria?

Reverse
transcriptase
can be used to
obtain coding
regions without
introns.

Fig 20.14

After RT, PCR will
amplify the gene or DNA

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

Fig 20.2

Cutting the
plasmid and insert
with the same
restriction enzyme
makes matching
sticky ends

A typical
bacterial plasmid
used for genetic
engineering

Using sticky ends to add DNA to a bacterial plasmid

Fig 20.2

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 self
-
ligation

Fig 20.8

Transformation of bacteria can happen via
several different methods.

Fig 7.2

Bacteria can take up DNA from the environment

Fig 20.8

Transformation of bacteria can happen via
several different methods all involving
perturbing the bacterial membrane:


Electroporation


Heat shock


Osmotic Stress

How can you know which bacteria have been
transformed, and whether they have the insert?

Figure 20
-
5

Fig 20.5

Resistance genes allow
bacteria with the
plasmid to be selected.

Bacteria with the resistance
gene will survive when
grown in the presence of
antibiotic

Figure 20
-
5

Fig 20.5

Is the insert present?

Plasmids with the MCS
in the lacZ gene can be
used for blue/white
screening…

A typical
bacterial plasmid
used for genetic
engineering

Figure 20
-
5

Fig 20.5

Intact lacZ makes a
blue color when
expressed and provided
X
-
galactose

Figure 20
-
5

Fig 20.5

When the lacZ gene is
disrupted, the bacteria
appear white

Blue/white
screening:

Transformed
bacteria plated on
antibiotic and X
-
gal plates.

Each colony
represents millions
of clones of one
transformed cell.

Successful transformation
will grow a colony of
genetically modified
bacteria

Fig 20.4

Inserting a gene into a
bacterial plasmid

RT and/or
PCR