Team ___ or initials +DNA LB amp Period __ Team

twoeggfinnishΒιοτεχνολογία

14 Δεκ 2012 (πριν από 4 χρόνια και 10 μήνες)

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Making Cells Glow:

Bacterial
Transformation with
pGLO

Plasmid DNA



BACTERIAL TRANSFORMATION,

GENETIC ENGINEERING,

AND RECOMBINANT PROTEINS

2

Essential Components of

Genetic Engineering

3

bacterial

animal

plant

HOST
CELLS

with their
own
genomic
DNA

A VECTOR

a plasmid or virus DNA used to


assemble the recombinant
construct


maintain it in its temporary and
permanent host cells


introduce the
transgene

into
cells


ensure expression of the
transgene

in the new host cell

A TRANSGENE

intended to give the host
cell or organism new or
altered traits.

Plasmids are frequently used as vectors.

Extrachromosomal

bacterial DNAs

Small

4000
bp
; compared to bacterial
chromosome of ~4 million base
pairs

Easy to work with

Can be removed, altered, then
returned to cells.

Replicate independently of
bacterial chromosome

Single or multiple copies per cell

Discovered in nature as a source
of antibiotic resistance

4

http://commons.wikimedia.org/wiki/File:Plasmid_episome.png

plasmids

host
chromosome

Some plasmids integrate
into the host genome

Origin of Replication

ensures replication of DNA in a host cell

Selectable Marker

allows for selection of
transformants
;
usually confers antibiotic resistance

Reporter Gene

gene whose phenotype changes depending
on whether a foreign
transgene

has been
inserted into the plasmid

Promoter(s)

promotes transcription of selectable marker
and
transgene
/reporter gene

Polylinker

or Multiple Cloning Site (MCS)

series of closely spaced, unique restriction
sites at which the plasmid can be cut
(
linearized
) to allow insertion (ligation) of
the
transgene

into the plasmid

5

polylinker

origin of
replication

s
electable
marker

reporter
gene

promoters

Features of a Typical

Cloning Vector

Ampicillin

Beta
-
Lactamase

bla
:

a common selectable marker

An antibiotic

Prevents the growth of bacteria by
inhibiting an enzyme that is
needed for building new cell wall
peptidoglycan

Chemical structure based on a Beta
-
lactam

ring

Beta
-
lactam

antibiotics include
peni
cillin
s

(
amoxycillin
) and
ceph
alosporins

An enzyme

Chemically breaks the beta
-
lactam

ring, inactivating the enzyme

The
bla

gene

Encodes the beta
-
lactamase

enzyme

Makes bacteria resistant to
ampicillin

(
amp
r
)

6

Prokaryotes

Eukaryotes

Issues in Moving
Transgenes


Between Hosts

Bacteria

Simple cell structures

No nucleus; DNA spread throughout
the cell

Simple gene structures

Protein
-
coding DNA sequence
(open
reading frame, or ORF) is contiguous

No machinery for RNA splicing




Simple promoters

Fewer transcription
factor proteins

Fungi,
Protists
, Plants, Animals

Complex cell structures

Internal membrane
-
enclosed
organelles, including a nucleus

Complex gene structures

Protein
-
coding DNA sequence (
exons
)
is interrupted by non
-
coding
sequences
introns

Requires RNA splicing to convert pre
-
mRNA (primary transcript;
exons

+
introns
) into mRNA (
exons
)

Complex promoters

Many transcription factor proteins

7

Eukaryotic Gene Structure

8

http://commons.wikimedia.org/wiki/File:DNA_exons_introns.gif

cDNA

Cloning of Eukaryotic Genes into
Prokaryotic Hosts

Since eukaryotic genes
have
introns

that
prokaryotic cells can’t
remove, a
cDNA

transgene

is created from
a DNA copy of the mRNA
with
introns

removed.

cDNA
: complementary
DNA

Transgene

must be
attached to a prokaryotic
promoter to ensure
transcription in new
bacterial host.

9

Regulation of Transcription in Prokaryotes
at the P
ara

and
P
lac

Promoters

In the
absence of inducer
(lactose or
arabinose
),
transcription is turned OFF


Repressor protein binds to the
operator, blocking the RNA
polymerase from the
promoter.

In the
presence of inducer
(lactose or
arabinose
),
transcription is turned ON

Inducer binds to the repressor
protein, causing a change in its
shape. The repressor falls off
the operator, allowing RNA
polymerase to bind to the
promoter and transcribe the
gene.

10

Recombinant Proteins

Proteins that are produced through genetic
engineering.

Encoded by the introduced
transgene
.

Produced upon the
transgene’s

transcription and
translation.

Can be purified from the transgenic cells or
organisms.

Can be produced in much higher quantities that
protein available from natural sources.

11

Medicine

Industry & Consumer
Products

Examples of Recombinant Proteins

Human insulin
(1982): Used to treat diabetes.

rHGH

or human growth hormone
(1985): Stimulates
growth (height) and development of muscle.

Cadaver
-
derived natural HGH transferred
Creutzfeldt
-
Jacob
disease (1985)

rBST

or bovine
somatotropin

(1994): Stimulates milk
production in cows

R
equired or permissible labeling of
rBST

milk or non
-
rBST

milk is
debated

EPO or
erythropoeitin
: Stimulates creation of red blood
cells.

Used to treat anemia in cancer chemotherapy patients. Common
blood doping agent for athletes.

tPA

or tissue
plasminogen

activator
: Enzyme given to
heart attack patients to dissolve blood clots blocking
arteries.

Factor VIII
: Blood clotting factor missing in hemophiliacs

In Laundry Detergents

Protease

for proteins,
lipases

for greases, and
amylases

for carbohydrates

Amylases and
Maltases

For production of high fructose corn syrup from corn
starch

Cellulases

and
Ligninases

Enzymes that digest cellulose into sugars to be fermented
in ethanol for
biofuels


Pectinases

Clarify fruit juices

Rennin

Used in cheese production

12

SESSION 1:

TRANSFORMING THE BACTERIA

Mixing bacterial cells and DNA under
transformation conditions.

Introduces DNA into cells.

13

The
pGLO


Plasmid

14

bla

gene:

a s
electable

marker;

encodes
beta
-
lactamase

enzyme; confers
amp
r

phenotype

gfp

gene:

a
transgene
;

encodes Green
Fluorescent
Protein (GFP);
confers glowing
phenotype

P
ara

promoter
:

allows
transcription of
the
gfp

gene
when cells are
treated with
arabinose

Origin of
replication

araC

gene:

encodes the
repressor protein
that blocks
transcription at
P
ara

promoter in
absence of
arabinose

Label Your Transformation Culture Tubes

Use a lab marker to label two 15ml
round
-
bottom culture tubes:

-
DNA
& your initials

+DNA
& your initials

Place these tubes in your ice cup to
chill.

It is
very

important that the
transformation reactions be keep cold.
Don’t handle these tubes or have them
out of the ice for more than a few
seconds at a time.

15

-
DNA initials

+DNA initials

Making the Transformation Mixtures

To
each
tube, add
100µl

of competent
E.
coli
cells
.

Provided in a
microtube

in your ice cup

Pipet

slowly (the cells are fragile)

Carefully deposit the drop of cells to the very bottom of
the tube.

Keep tubes on ice.

Promptly replace the snap
-
on caps to avoid
contamination by bacteria and fungi in the air.

Don’t forget: always use a fresh
pipet

tip each time!

To the

DNA

tube, add
10µl

of
TE Buffer

directly to the
drop of cells

To the
+DNA

tube, add
10µl

of 5ng/µl
pGLO

plasmid
DNA

directly to the drop of cells.

16

-
DNA initials

+DNA initials

-
DNA initials

+DNA initials

Cold
-
Incubating the Transformation
Mixtures

Gently tap the bottom of each tube to gently
mix the cells and solutions.

Incubate on ice for 15 minutes.

During this time, DNA becomes attached to the
outer surface of the cells.

17

Heat
-
Shocking the Transformation
Mixtures

Bring your ice cup with the two culture tubes to the
42
°
C heat block.

Quickly place your pair of tubes into the heat block.
Note the time.

After
exactly 45 seconds
, quickly remove your pair
of tubes and immediately place them back in your
ice cup for at least one minute.

This “heat
-
shock” step opens pores in the cell’s
membranes, allowing the DNA to enter
some

cells. The
heat shock requires instantaneous transitions between
cold to hot to cold.

18

Initial Cell Culture:

Recovery and
bla

Gene Expression

Add 800µl of LB broth to each culture tube.

Don’t forget: always use a fresh
pipet

tip
each time!

Replace the caps promptly to avoid
contamination.

Tap the bottom of the tube to mix.

Place the tubes into the foam adapter
mounted on a vortex mixer.

Your samples will be agitated at room
temperature for about 45 minutes. This gives
the new genes (on the plasmid DNA)
introduced into the cells time to be
transcribed and translated into proteins.

19

-
DNA initials

+DNA initials

Teacher Note

After about a class period of incubation (40
-
50
minutes), transfer the tubes to a lab refrigerator
(
without food!
) for overnight storage.

20

SESSION 2:

SPREAD
-
PLATING THE
TRANSFORMATION CULTURES

Growing the transformation cultures on
non
-
selective, selective, and indicator
plates.

21

Culture Media

LB

Luria
-
Bertani

medium: a rich medium that provides
a complete mixture of nutrients (sugars, amino
acids) and vitamins in which bacteria can grow.

agar

a substance added to media that makes it semi
-
solid

22

Culture Media Additives

amp:
ampicillin

an antibiotic that kills bacteria,
except those cells that contain
genes that provide resistance
(such as the beta
-
lactamase

or
bla

gene, sometimes called an
ampicillin
-
resistance or
amp
r

gene)

selective medium

a growth medium that causes the
death, or prevent the growth, of
some cells but not others

ara
:
arabinose

a sugar that induces transcription
of a gene by removing the
repressor protein from the
gene’s specific “
ara
” promoter

indicator medium

a growth medium that causes
some cells to appear differently
than other cells, indicating the
presence or absence of certain
traits

23

Why Grow Transformation Cultures On
Selective Media?

The transformation process is
very
inefficient.

Only a tiny fraction of the cells actually take up the DNA.

We face a “finding a needle in a haystack” problem.

How do we detect, and obtain, only the cells that have
been successfully transformed (the “
transformants
”)?

“Burn down the haystack!”

Kill off all the non
-
transformants

on selective media.

Cells lacking the
pGLO

plasmid will lack its
bla

gene, and
thus will be sensitive to
ampicillin
.

24

To possess a trait, you need to both
possess

the gene and
express

it.

The
gfp

gene encodes the green
fluorescent protein (
GFP
) from
the bioluminescent jellyfish
Aequorea

victoria
.

For a cell to have GFP, it must
transcribe

and
translate

the
gfp

gene.

Arabinose

induces the
transcription of the
gfp

gene.

The
gfp

gene is
expressed

when
transformed cells are treated
with
arabinose
.

25

http://en.wikipedia.org/wiki/File:Aequorea_victoria.jpg

http://en.wikipedia.org/wiki/File:GFP_structure.png

Labeling the Plates

Period __

Team ___

or initials

Period __

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or initials

Period __

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or initials

Period __

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or initials

Period __

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or initials

Period __

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or initials

Non
-
selective
plate

Selective
plate

Selective &
Indicator
plate

26

Period __

Team ___

or initials

Period __

Team ___

or initials

Period __

Team ___

or initials

Period __

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or initials

Period __

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or initials

Period __

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or initials

Spread
-
Plating the

Transformation Cultures

-
DNA initials

+DNA initials

200µl

200µl

27

Spread
-
Plating

Your foil packet contains two sterile yellow spreaders. Feel the foil
packet and find the end shaped like a triangle. Carefully open the foil
at the stick (not the triangle) end, keeping the triangle ends covered
with foil. Keep the spreader in the opened pack for now.

Turn your three

DNA

plates over (agar side on bottom) and apply
200µl of your

DNA

culture to
each

of the three

DNA
plates:
LB, LB
amp, and LB amp
ara
.
Use a fresh
pipet

tip each time.

Remove one spreader from the pack (keep the other spreader
covered) and use it to
gently

spread the liquid across the entire
surface of each plate, turning the plate as you spread.
Don’t press too
hard, or the agar will tear.
Place the used spreader in the collection
bin.

Repeat using the other spreader to apply the
+DNA

culture to each of
the three
+DNA
plates.

28

Incubating Your Cultures

Allow your plates to sit, agar side down, for a
few minutes to allow the liquid to absorb into
the agar.

Tape your set of six plates together using
colored lab tape.

Label the tape with your class period.

Place you set of six plates into the 37
°
C
incubator for an overnight incubation.

29

Predict Whether Cells Will Grow On Each
Plate, and What They Will Look Like

-

DNA

+DNA

Grow?

Lawn or
Colonies?

Glow
under UV
light?

Grow?

Lawn or
Colonies?

Glow
under UV
light?

LB

LB

amp

LB

amp
ara

30

Teacher Note

After overnight incubation, if the students will
not be observing their results the following day,
wrap the plates in
parafilm

and store them in a
refrigerator (
with no food!
).

31

SESSION 3:

INTERPRETING RESULTS

Examining for evidence of transformation
and recombinant gene expression.

32

Imagining Mind

Thinking Mind

Use Your “Two Minds”

Imagines what’s possible

Finds all alternatives

Decides what’s real

true or false

Eliminates alternatives

33

Scientific Thinking is Critical Thinking

E

A

C

+

=

Evidence

“Facts we SEE”

Assumptions

“Things we THINK”

Conclusions

“Claims we MAKE”


Observations


Data


Results


Materials


Procedures


Experimental
Design


CONTROLS

34

Words of Wisdom from Sherlock Holmes

“It is an old maxim of mine that when you have
excluded the impossible, whatever remains,
however improbable, must be the truth.”

35

DESIGNING AN EXPERIMENT:

EXPERIMENTAL VARIABLES


Manipulated Variables


Controlled Variables


Responding Variables


Uncontrolled Variables

36

Manipulated Variables

Also called the
Independent Variable
.

The condition or treatment that is changed or
manipulated during the experiment.

Each sample is subjected to different conditions
for the manipulated variable: treatment,
amount, time, duration, etc.

The manipulated variable is the "cause" for
which we wish to identify an "effect".

37

Controlled Variables

Conditions and treatments that are identical for
all samples within the experiment

Conditions that are to be ruled out as affecting
the outcome.

38

Responding Variables

Also called the
Dependent Variable
.

The properties to be observed or measured.

The "effect(s)" associated with changes in the
manipulated variable.

39

Uncontrolled Variables

Factors which may impact experimental samples
or subjects differently, resulting in effects that
are not due to the manipulated variable.

Experimenter error

Bias

Environmental conditions

Non
-
random sampling

40

ANALYSIS OF RESULTS

41

Arrange your plates like this.

Period __

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or initials

Period __

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or initials

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or initials

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or initials

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or initials

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or initials

42

What do you conclude from THIS plate ALONE?

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or initials

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or initials

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or initials

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or initials

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or initials

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or initials

What ELSE might you conclude?

Critical
Thinking
involves
identifying
and
considering
ALL
alternatives!

Negative
results have
no meaning
EXCEPT

in
comparison
to a POSITIVE
CONTROL.

43

What do you conclude from THIS plate ALONE?

Period __

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or initials

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or initials

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or initials

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or initials

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or initials

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or initials

44

What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?

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or initials

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or initials

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or initials

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or initials

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or initials

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or initials

45

What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?

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or initials

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or initials

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or initials

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or initials

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or initials

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or initials

46

What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?

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or initials

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or initials

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or initials

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or initials

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or initials

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or initials

47

Examining for Production of

Green Fluorescent Protein

Turn out the room lights.

Hold the UV lamp over your plates.

Do not

look directly into the UV lamp.

Record which of your plates have colonies that
glow green.

48

What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?

Period __

Team ___

or initials

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or initials

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or initials

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or initials

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or initials

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or initials

49

GOOD EXPERIMENTAL DESIGN


Seeks to control variables.


Confirms all of our assumptions about
materials and procedures.


Allows us to conclude a clear

CAUSE
-
and
-
EFFECT relationship between
the MANIPULATED VARIABLE and the
RESPONDING VARIABLE.

50

Natural Light

UV Light

Sample Results

51

What Questions Do YOU Have?

52