Genetic Engineering Laboratory Announcements One-Minute Paper ...

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11 Δεκ 2012 (πριν από 4 χρόνια και 9 μήνες)

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1
Genetic Engineering Laboratory

Constructing a Phage
λ
Library
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
2
Announcements

Exam Short Answer

Back today; average = 22.2/25

Presentation marks

Back today; average = 8.8/10

Assignment marks

Back next Tuesday

Quiz #2 - next Tuesday

Lab website

Good genetic engineering links
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
3

Quiz #1

Fair, but too long

Participation points

Unfair to require questions

Alternatives?
One-Minute Paper Feedback
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
4
Genetic Engineering

All life contains similar genetic machinery

This similarity allows splicing together of
unrelated genes

Amplify gene of interest

Modify genes

Move genes

Study properties/defects of genes
2
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
5
Laboratory Goals

To learn important concepts and jargon in genetic
engineering

To perform hands-on cloning

To understand and implement the selection/
screening process

To learn the
‘sterile technique

Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
6

Double stranded circular DNA

chromosome


Occurs naturally, though modified for our uses

Similar to mtDNA - relatively short, few genes

However,
not
essential to organism (normally)

Replicates independently of the host

Can have many (many) copies per cell

A plasmid is a
vector
in the sense that it can carry
DNA from one organism to another
Plasmids
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
7
Restriction Endonucleases

Cut double-stranded DNA fragments at
specific
nucleotide sequences (4-6 bases)

Also called

restriction enzymes


Break

phosphodiester
bonds between nucleotides

Different enzymes recognize different patterns

Some cleave

cleanly

and leave
“blunt ends


Others make
“staggered cuts

and leave

sticky
ends

- overhanging of nucleotides

Type of end left is important in
ligation
stage
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
8
Example Enzyme
A
G
T
CC
A
G
A
GG
A
T
CC
T
A
C
A
GG
A
T
A
G
A
C
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AA
T
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G
A
CC
A
T
C
A
GG
T
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CC
T
A
GG
A
T
G
T
CC
T
A
T
C
T
G
C
TT
A
T
C
T
GG
T
BamHI
“sticky ends

A
G
T
CC
A
G
A
G G
A
T
CC
T
A
C
A
GG
A
T
A
G
A
C
G
AA
T
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G
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C
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GG
T
C
T
CC
T
A
G G
A
T
G
T
CC
T
A
T
C
T
G
C
TT
A
T
C
3
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
9

Forms
phosphodiester bonds between DNA fragments
via the enzyme DNA Ligase

Opposite function of restriction enzymes

Only
joins ends that are compatible

Any
Blunt with any Blunt

Specific
Sticky with specific Sticky

Important to note that ligation is

blind


Will join
any
compatible ends (
very
inefficient)
Ligation
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
10
Transformation

Most cells do
not
readily take up DNA

Have nucleases and impermeable membranes

Griffith was lucky

Many cells can be made transformable

Treatment with MgCl
2
and CaCl
2

Transformation efficiency is
very
low

Only a few insertions will be
‘correct


Only a few cells pick up plasmids
nucleases
membrane
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
11
Transformation
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
12
Cloning

The process of putting a gene of interest into a plasmid
for the purpose of making many copies of the gene
within an organism

Also called Genetic Engineering or Recombinant DNA
technology

Involves
cleaving

both
plasmid
and
target DNA with
restriction enzymes, followed by
ligation
and
transformation (putting plasmid in a cell)

Must
use compatible enzymes on both fragments
*
4
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
13

Required to determine
which
cells (if any) contain the
desired DNA fragment

Use genes present on plasmid but not in native cell

Selection
: determine which cells
contain
a plasmid

Screening
: from those above, determine which cells
contain a
recombinant
plasmid

Our strain of
E. coli
(DH5
α
) cells have their
lac
Z genes
‘knocked out


These cells will not be able to metabolize lactose
How can we use this for our purposes?
Selection/Screening
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
14
Plasmid for today - pGEM3z
Carries two genes:
1)
bla
- codes for
ß
-lactamase
- breaks down ampicillin
- allows bacteria to survive in a
normally lethal medium
-
use this gene for selection/screening purposes
- cells cannot survive exposure to ampicillin
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
15
Plasmid for today - pGEM3z
Carries two genes:
2)
lacZ
- metabolism of lactose
-
β
-galactosidase
- can break down

X-Gal

(similar
structure) to a blue product
- gene is induced by IPTG (see lab manual)
- also good for selection/screening
- cannot break down X-Gal without
lacZ
gene
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
16
Phage
λ

A bacteriophage, or

phage
’ is simply a virus that
infects bacteria

Has genes for invasion and reproduction

Read lab manual for further information

We will clone phage
λ
DNA into pGEM3z

Gene library - a collection of
various pieces of
genetic material that altogether provide a good
representation of the genome as a whole
IS A PHAGE ALIVE?
5
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
17
Cloning

Following cloning we
must check to see if we
were successful

Plasmids come with
genes we can use to
screen cells

i.e. genes that allow
growth in a normally
lethal medium
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
18
Today

We will begin our three part laboratory by
constructing a phage
λ
gene library

Vector and phage DNA have been pre-cut

You must:

Perform ligation

Transform recombinant plasmids

Set up selection/screening process
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
19
Protocol For Today
1) Ligation (already done)

Incubate
45
minutes at room temperature

Exams/presentation marks back
2) Add competent cells

Incubate on ice for
20
minutes

Learn sterile technique
3) Incubate at 42
˚C for
2
minutes
4) Plate 100

L
cells
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
20
Sterile Technique

Introduction of foreign DNA will
compromise our results

Therefore, everything we use must be
first sterilized

I will walk you through this, plus we
will purposely contaminate a control
plate to the observe results
6
Biology
162
Laboratory
#4
-
Genetic
Engineering
Lab
Joseph
W.
Brown
-
February
7,
2006
21

Next week:

Continue with Genetic
Engineering laboratory

Quiz #2 (Tuesday)

Assignment #2

Questions 1-5 on page #60

Due:
February 16 (Discussion)

Discussion: more genetic engineering

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