Lab Analysis

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

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LAB



Genetic
Transformation

Introduction

Part 1


Background


Genetic engineering is a process that enables scientists to put a desired gene in
to a bacterial plasmid.
The plasmid and gene must be prepared so that they can be joined together to form a new
,

recombinant
plasmid.

If both the gene and the plasmid are cut with restriction enzymes that leave complementary
sticky ends, these ends may the
n be joined by
DNA
ligase to make a new plasmid. Since this plasmid
has DNA from 2 different organisms, it is considered to have
recombinant DNA
.




In this lab
, you will perform the

next step known as
genetic

transformation
.

During
transformation
,

a
bac
terial cell takes in a recombinant plasmid containing a gene of interest that has been combined with
the bacterial DNA.
Remember that a gene is a piece of DNA that provides the instructions for making
a protein. This protein gives an organism a particular
trait. Genetic transformation is used in many
areas of biotechnology. In
agriculture
, genes coding

for traits such as frost, pest,

or spoilage resistance
can be genetically transformed into plants

(aka genetically modified foods)
. In
bioremediation
, bacter
ia
can be genetically transformed with genes enabling them to digest oil spills. In
medicine
, diseases
caused by defective genes are beginning to be treated by gene therapy; that is, by genetically
transforming a sick person's cells with healthy copies of
the defective gene that causes the disease.


Part 2


Lab Preparation


In this lab, you will transform bacterial cells

by moving genes from one
organism to another with the aid of a plasmid. In addition to one large
chromosome, bac
teria naturally contain one or more small circular pieces of
DNA called
plasmids
. You will be inserting a
recombinant

plasmid, called a
pGLO plasmid
, into the normal strain of E.coli bacteria
l cells
. The pGLO
plasmid DNA already has two unique genes insert
ed within it.


The first gene that has been added to the pGLO plasmid is the GFP gene,
which codes for the production of
Green Fluorescent Protein (GFP)
. The
real
-
life source of this gene is the bioluminescent jel
lyfish

Aequorea victoria
.
Green Fluorescen
t Protein causes the jellyfish to fluoresce and glow in the
dark! Following the transformation procedure, if our normal strain of E.coli
bacteria express their newly acquired jellyfish GFP gene and produce the
fluorescent protein, they will glow a brillian
t green color under
UV

light.


The second gene that has been added to the pGLO plasmid is the beta
-
lactamase gene, which codes for
bacterial resistance to the antibiotic
ampicillin
. Ampicillin

is an antibiotic that kills various bac
terial
strains.
However,

if bacteria
l cells

have a certain gene resistant to ampicillin (
the
beta
-
lactamase gene),
then exposure to the drug ampicillin does not kill the bacteria.


As a result, normal strain E.coli plasmid DNA has
gone through genetic recombination

to include
b
oth
the GFP gene sequence and the beta
-
lactamase gene sequence

(like you did in the previous lab!)
.
However, there is one last catch to see the bacteria grow & glow! The pGLO plasmid also requires a
special gene regulation system

to control the expression
of the fluorescent protein in transformed
cells. The gene for GFP can be switched on in transformed cells by adding the sugar
arabinose

to the
bacterial cells' nutrient medium. Transformed cells will appear white on plates not containing
arabinose, and fluorescent green when arabinose is included in the nutrient agar. In addition, one can
test for bacterial cells that have been transforme
d with pGLO DNA by growing them on plates with
ampicillin.

Advance Preparation

Please answer the following questions in complete sentences
after

reading BOTH the intro
duction and
the procedure.

Your preparation for this lab will determine your understandin
g of how these bacteria
will grow and glow.

Part 1
-

Background

1.

What is
genetic transformation
?

2.

To genetically transform an entire organism, you must insert the new gene in
to every cell in
the organism.
Which organism is better suited for total g
enetic transformation:
a
prokaryote, or a eukaryote? Why?

3.

Scientists often want to know if a genetically transformed organism can pass its new traits
to its off
spring and future generations. Why,
then, are bacteria most frequently used in these
genetic tra
nsformation investigations?

(HINT: Recall the reasons Mendel chose pea plants!)

4.

What are some additional
uses of genetic transformation?
Please describe.


Part 2


Lab Preparation

5.

What protein does the
GFP

gene

code for? In what organism can this gene be found?

6.

What protein does the
beta
-
lactamase gene

code for? Why would it be beneficial for
bacteria
l cells

to have the beta
-
lactamase gene?

7.

How is the GFP gene “switched on” in transformed bacterial cells?

8.

What w
ill bacteria that have the
pGLO plasmid

(containing the GFP and beta
-
lactamase
genes)

be able to do at the end of the lab?


Hypothesis

After having read the introduction and the lab procedure
carefully,
observe the four labeled agar plates
below.



“LB” sta
nds for Luria Broth, which contains the bacteria’s nutrients to grow



“amp” stands for ampicillin, an antibiotic that kills bacteria



“ara” stands for arabinose, the sugar that “switches on” the GFP gene









Based on what you know about the E.coli bacteria containing the pGLO plasmid (+pGLO),
predict
what you think will occur in the lab:


a)

On which plate(s) will the bacteria
grow
? Why?


b)

On which plate(s) will the bacteria
glow
? Why?



Plate 1

Plate 2

Plate 3

Plate 4

+pGLO:


tube with bacteria

containing
the
pGLO plasmid (GFP &
beta
-
lactamase genes)


-
pGLO:


tube with normal,

unaltered bacteria

Procedure: Day One

1.

Label one closed reaction tube “+ pGLO” and another “
-

pGLO” indicating which bacteria
you will add the pGLO plasmid to, and which will remain normal.












2.

One at a time, open each tube and use a pipette to transfer 250 microliters (µL) of
Transformation Solution (CaCl
2
) into each tube.



3.

Obtain ice in the cup provided. Then place the tubes on ice.

4.

Use a sterile loop to pick up one single colony of bacteria from your starter plate. Open up
the “+ pGLO” tube and immerse the loop into the Transformation Solution at the bottom of
the tube. Spin the loop until the entire colony comes off the loop. Clo
se the top and “finger
flick” the tube to disperse the clump of bacteria until you can see no floating clumps. Place
the tube back on ice! Using a new sterile loop, repeat for the “
-

pGLO” tube.














5.

I
mmerse a new sterile loop into the plasmid
DNA stock tube, which your instructor has.
Withdraw a loopful. There should be a film of plasmid solution across the ring.
Mix the
loopful into the cell suspension of the “+ pGLO” tube. Close the tube and return it to the
rack on ice. Also close the “


pGLO” tube.
Do not add modified plasmid DNA to the


pGLO tube.

Why not?







6.

Incubate the tubes on ice for 10 minutes. While the tubes are sitting on ice, label your four
agar plates on the

bottom

(not the lid) as follows. Then work on the Genetic
Transformation Review handout provided by your instructor as you wait.






7.

Heat shock.

Take your ice container and tubes to the 42 ºC water bath. Put BOTH the (+)
and (

) tubes into the rack in the water bath for
exactly 50 seconds
.

8.

After the 50 seconds, immediately place both tubes back on ice. For the best
transformation results, the c
hange from the ice to 42 ºC and then back to the ice must be
rapid!







9.

Incubate tubes on ice for
at least 2 minutes.

10.

Remove the tubes from the ice and place in the foam rack on the bench top. Open the “+
pGLO” tube and, using
a new sterile

pipette, ad
d 250 µl of LB nutrient broth to the tube
and reclose it. Repeat with a
new sterile pipette

for the “


pGLO” tube. Incubate the tubes
for 10 minutes at room temperature (i.e. on your lab table).









11.

Tap the closed tubes with your finger to mix.
Using a new sterile pipette for each tube,
pipette 100 µl of the transformation and control suspensions onto the appropriate nutrient
agar plates.



















12.

Using a new sterile loop for each plate,

spread the suspensions evenly around the surface
of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and
forth across the plate surface.
Do not press too deep into the agar!


13.

Stack your plates
upside down

and tape them
together, as shown in the picture below. Put
your group name and class period on the tape and place the plates in the tray at the center
lab station. Your instructor will put them in a 37 °C incubator until next class.



Clean up



Dispose of
all
plastic l
oops (and anything that has touched bacteria) in the bleach bucket.



Wash your hands with soap and
warm
water.

Procedure: Day Two

1.

Observe each of the four plates under normal room lighting. First, sketch the results
observed on all 4 plates. Then record
your observations in the data table.

2.

Observe each of the four plates under the ultraviolet light. Record your observations.

3.

Once your observations are complete, dispose of
all

of the plates in the bleach container.


Data


Lab
Analysis

1.

On which plates was
genetic
transformation

a success? Explain your choices.

2.

Wha
t is meant by a control plate?
Identify the

two control
plates
in this lab and discuss
their
purpose.

3.

Which of the plates resulted in bacterial

growth?
Discuss why

for

each plate.

4.

Which of the plates
had NO
bacterial

growth?
Discuss why

for each plate.

5.

Which of the plates
with growth

resulted in bacteria that produced a green glow under UV
light?
Explain why.

6.

Which
of the plates
with growth

resulted in bacteria that
did not glow? Why

not
?

7.

In biology, we talk about
Consistency and Change
as the evolution of organisms into
differrent forms, while maintaining their defining characteristics. How does this lab
illustrate the BioTheme
Consistency and Change?


Table 1. Final Transformation Data fo
r Experimental Plates

Plate

Relative Size
of Colonies

Number of
Colonies

Color

(in normal light)

Color

(under UV light)

Transformation
Success (yes/no)


+pGLO

LB/amp









+pGLO

LB/amp/ara









pGLO

LB/amp









pGLO

LB