Lab 5 Bacterial Transformation

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

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1

May 31, 2007


Genetic Engineering: Bacterial Transformation



What are bacteria?



Bacteria are single
-
celled microorganisms with a relatively simple cell structure. They do
not contain a nucleus with linear chromosomes. Instead, bacteria have a singl
e, large, circular
chromosome whose genes are generally essential to the life and reproduction of the bacterium.
They also
may

contain smaller, circular pieces of DNA called
plasmids
. The genes on plasmids
usually are not essential to the life and reprod
uction of the cell. Instead, the genes on plasmids
provide some advantage to the cells under specific environmental circumstances. Many strains of
bacteria pass plasmids from one bacterial cell to another. There are a variety of mechanisms by
which bacte
ria exchange plasmids, one of which, transformation, will be performed in this
exercise.


What is transformation?


Bacterial transformation is a process whereby cell
-
free DNA (found outside of bacterial
cell structures in the surrounding solution) is absor
bed by a living bacterial cell. When this DNA
uptake occurs, the genes may be expressed, i.e., new proteins synthesized. This will alter the
traits of the bacterium.


Transformation is rare in nature; most bacteria do not possess the natural ability to u
ptake
and express cell
-
free, foreign DNA. However, in 1970, M. Mandel and A. Higa discovered
certain factors which greatly increase the efficiency of transformation in the lab: 1) treatment
with a cold solution of calcium chloride, 2) a brief "heat shock"

at 42◦C, 3) performing the
experiment when the cells are growing very rapidly, and 4) transforming with
plasmids
. Despite
these advances, the exact mechanism of DNA uptake is still unclear.


Lab Objectives


In this experiment, students will transform t
he bacterium
E. coli

with a gene that codes for
Green Fluorescent Protein (GFP). The real
-
life source of this gene is the bioluminescent
jellyfish
Aequorea victoria.
GFP causes the jellyfish to fluoresce and glow in the dark.
Following the transformatio
n procedure, the bacteria express their newly acquired jellyfish
gene and produce the fluorescent protein causing them to glow a brilliant green color under
ultraviolet light.


2

The unique
p
GLO plasmid will be utilized in this lab. Two genes with identifia
ble phenotypes
are on this plasmid. The
E. coli

strain has neither of these genes.


1)

GFP gene



codes for Green Fluorescent Protein. When bacteria that

have been transformed are grown in the presence of the sugar arabinose,

the GFP gene is “turned on” and

the bacteria glow under U.V. light.

2)

bla gene


codes for the protein
beta
-
lactamase, which provides resistance to
the antibiotic ampicillin.




A fluorescent green colony phenotype is easy to recognize, but how will we know if the
bla

gene also was incorp
orated? Agar containing ampicillin will be our
selective

medium
. Only
transformed cells with the
bla

gene, and, therefore,
ampicillin resistant
, will grow. The
untransformed original
E. coli

strain will not survive and form colonies on this type of medi
a.



ABSOLUTELY NO FOOD OR DRINK IS ALLOWED IN THE LAB.


Gloves, aprons, and safety glasses must be worn at all times.


Dispose of pipets as instructed.


Dispose of all waste in biohazard bags.


Begin by cleaning the lab benches with the cleaning solution
and washing your hands.


Materials:
per lab bench


1 tube containing 3

ml calcium chloride solution in a 500

ml beaker containing


chipped ice.

E. coli

culture on agar

1 vial of
p
GLO plasmid (may share among all groups)

1 vial or tube containing 3

m
l broth

2 sterile 8

ml test tubes with cap

Three 1

ml pipets, pipet pump (blue)

Water bath at 42

C containing a test tube rack

5 disposable inoculating loops

2 plain
LB agar

plates

2
LB/
Amp
agar
plates

2
LB/A
mp/Ara
agar
plates

Gloves, safety glasses
, sh
arpie markers

Biohazard bag

Ethyl alcohol in jars with lids and Bactispreaders (one/lab bench), Bunsen burners,
Stikers


3

Students will suspend

untransformed

E. coli

cells in two tubes containing cold calcium
chloride solutions. One of these tubes will serv
e as our
control
; plasmid will not be added to the
control tube. In the other tube, the
experimental

tube,
p
GLO plasmid will be added. Both tubes
will then be incubated on ice for 10 minutes. A brief "heat shock" at 42

C will follow. The cells
are then

cooled and broth added. Samples will be plated on three types of media: 1) plain
LB
agar, 2)
LB
agar with ampicillin (Amp agar), and 3)
LB
Amp agar that also contains arabinose
(
LB/
Amp/Ara agar). After incubation, bacterial growth and the appearance of
fluorescent green
colonies under U.V. light will be checked.


Day 1


Procedure:



1. Label one sterile 8

ml tube "+
p
GLO
". Label another sterile 8

ml tube "
-

p
GLO
".



2. Using a sterile pipet, add 0.25

ml of ice
-
cold calcium chloride to each tube.



3. Immediately, place both tubes on ice.



4. IN THIS STEP, BE CAREFUL NOT TO TRANSFER ANY AGAR FROM THE PLATE TO
THE TUBE. AGAR CAN INHIBIT THE TRANSFORMATION PROCESS.

Do not puncture
the agar; carefully swipe the
surface

of the agar to remove bacteri
a!


-
Using an inoculating loop
,
transfer one or two large colonies of
E. coli

to the
+
p
GLO

tube
containing calcium chloride.


-
IMMERSE THE LOOP
with the bacteria
IN THE CALCIUM CHLORIDE SOLUTION, AND
VIGOROUSLY TAP AGAINST WALL OF TUBE TO DISLODGE CELL MA
SS.


-
MAKE SURE THAT THE CELL MASS IS NO LONGER ON THE LOOP.




5.
IMMEDIATELY

suspend cells in the
+
p
GLO

tube by CAREFULLY stirring the solution with
the loop. Do not produce bubbles or splash the solution up against the walls of the tube. Hold
the tub
e up to the light to be sure the solution is uniform. No visible clumps of cells should
remain in the tube.



6. Return the
+
p
GLO

tube to the ice.


4


7. Again, use the loop to transfer another
bacterial mass

from the plate to the

-

p
GLO

tube and
suspend
as described in step 5. Return the
-

p
GLO

tube to the ice.



8. Both tubes should be on ice.



9. If possible, keep the cell suspension in the ice while performing this step. Use a clean,
inoculating loop to transfer ONE
loopful

of the
p
GLO
plasmid

solut
ion to the
+
p
GLO

tube.


The plasmid solution should form a "bubble" on the loop. Immerse the loop
directly into

the
E.
coli

cell suspension, and swish the loop
several times
to disperse the plasmid.


When finished, discard the plastic inoculating loo
p in the biohazard bag.


10. Incubate both tubes on ice for an additional 10 minutes. Go to Step 11 while the tubes are
incubating in the ice.


11. Observe the following plates and label if necessary.


a)
(LB/Amp)
Plate A

+
. This is an
experimental
plat
e containing transformed cells

from

the +
p
GLO tube
.


b)
(LB/Amp)
Plate B

-
. This is a
negative
control plate. It will NOT contain transformed
cells
, only the control cells (from the

p
GLO tube) that did not receive the plasmid
.


c)
(LB/Amp/Ara)
Plate C

+
.
This is an
experimental
plate containing
transformed cells

from the +
p
GLO tube.


d)
(LB/Amp/Ara)
Plate D

-
.
This is a
negative
control plate. It will NOT contain
transformed cells, only the control cells (from the

p
GLO tube) that did not receive the

plasmid.


e)
(LB)
Plate E

“experimental control
+
".
This is an
experimental
plate containing
transformed cells from the +
p
GLO tube.


f)
(LB)
Plate F

“control
-
".
This is a control plate. It will NO
T contain
transformed cells,

only the control cells (fr
om the

p
GLO
tube) that did not receive the
plasmid.


12. Following the 10 minute incubation on ice,
"heat shock"

the cells. Carry the tubes STILL
ON ICE to the 42

C water bath. Remove both tubes
directly from the ice and immediately
immerse

them in the
water bath for

50 seconds
.


5


13. Return both tubes directly to the ice for two minutes.


14. Add 0.25

ml of
broth

to each tube, using a new, sterile p
ipet with each tube. Gently tap the
bottom of the
tubes with finger to mix and set tubes in a test tube r
ack at

room temperature for
10 minutes
.


For Steps 15
-
18

(next page), follow the inoculation procedure in the illustration
below.



































IMPORTANT! Use different
pipets

for transferring +
p
GLO and

p
GL
O bacteria,


EXPERIMENTAL

CONTROL

+
p
GLO tube

E.coli + Plasmid (Transformed)

-

p
GLO tube

E. coli only (Untransformed)

Plate A

Amp

Plate B

Amp

Plate C

Amp/Ara

Plate D

Amp/Ara

Plate E

Plain
LB
agar

Plate F

Plain
LB
agar

0.1ml into
each

plate

0.1
ml into
each

plate


6

o
ne for the
+

p
GLO tube and a different one for the


p
GLO tube. DO NOT MIX THEM.
















15
. Observe the following precautions while transferring the bacteria to the plates.



Only lift the lids off of the plates when ready to trans
fer bacteria.


Do not set the plate
lids

on the bench; hold them.


Do not puncture the agar with the
pipet
.



Replace the lid as soon as you have transferred the solution.


16. See illustration on previous page: Pipet 0.1

ml of +
p
GLO
bacteria to the
SURFACE of the
agar (
DO

not to puncture the agar with the loop)

in

P
late

A.


17. Repeat step 16 with plates
C and E

(Transfer
0.1 ml

of bacteria from the same tube,
+
p
GLO, to plates C and E).


18.
Use a
different

pipet

to transfer
0.1 ml of
bacteria fro
m the
-

p
GLO

tube to

each of plates B, D, and F
.


19. Procedure for alcohol sterilization of glass bactispreaders :


a. Dip the glass bactispreader into the ethanol (in
jar
). DO NOT ALLOW THE
ETHANOL TO DRIP DOWN THE HANDLE OF THE BACTISPREADER!!!


b.

BRIEFLY pass the bactispreader through the flame.



c.

Immediately move the
flaming

bactispreader away from the flame and the
ethanol
.


d. Allow the flame to burn out. Cool for
15 seconds
.


e. Rub the bactispreader on an area of the agar
away

from th
e bacteria to cool.


f. Spr
ead the cells over the agar of P
late A.


g. Flame the bactispreader again.



20. Repeat step 19

for
the rest of the plates. Flame and cool between each plate and after you are
finished.


7




21
. Allow the plates to set for a few
minutes, then wrap each group’s plates together with tape

and label
.


22
. Place plates
inverted

in the 37

C incubator for
24
-
48 hours.


Expected Results:


1) Plate A:










2) Plate B:










3) Plate C:










4) Plate D:










5) Plate E:










6) Plate F:











Day 2


Observe the plates for growth under normal room lighting and under U.V. light

(Long Wave)
.


WEAR SAFETY GLASSES WHEN VIEWING UNDER U.V. LIGHT!







8

Name:







BIOL 117
Genes: The Foundations of Life Laboratory


Transf
ormation






1.
-
Actual Experimental

Results:



Plate A:















Plate B:












Plate C:












Plate D:













Plate E:












Plate F:












2. What are bacteria?
































3. Define transformation.









































4. List the two genes that were transferred in this lab AND their functions.





































9


5. Which plates should be compared to determine if any genetic transformation has occurred?
Why?



























































6. In detail, explain why we included Plates E and Plate F in the experiment?