pGreen Transformation - TeacherWeb

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

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Genetic Engineering

Lab: Bacterial Transformation with pGreen


Background:

Genetic engineering

or transgenic science

is a process that molecular biologists use to
insert a foreign piece of DNA into a host cell such as bacteria, yeast, animal or plant cells. A
biotechnology company formerly known as
Genentech

was the first to make human insulin from a
genetically transf
ormed

bacterial cell.
The gene for human insulin was isolated
and
“spliced” into

a
special circular piece of DNA called a plasmid
.
The plasmid is then inserted into the host bacterium,
by a technique called “heat shocking”.
The bacteria recognize and

express
the genes on th
e plasmid

into proteins.
Every time the cell divides (20 minutes) the offspring inherited the plasmid, making
them
clones.

This is how many human hormones, such as
insulin and human growth hormone (HGH
)
are

mass
produced
.

The
transformation lab that you will perform involves a plasmid called pGreen. It has two
genes on it. One for resistance to the antibiotic called
ampicillin.

This is a type of
genetic marker

to
let scientists know the bacteria have been transformed. The second gene is for bioluminescence that
will cause the bacteria to glow a green color in the dark. This
too serves as a
visual gen
etic.

The host
cell,
E.coli
MM294

is genetically designed

by

American geneticist Matthew Meselson. It was primarily
developed to be a competent host cell type to accept a foreign piece of DNA. It will not pose any
health risk to any organism. This cell is used worldwide in research institutions, universities, a
nd high
schools.


Purpose:

To genetically transform the
E. c
oli

MM294

bacterium
by inserted the pGreen plasmid.


Materials:

Groups of 4 students

Starter colony
plate

CaCl2 solution

Permanent
marker

Used tips/tube
container

Micro centrifuge

(optional)

1 LB only plate

LB broth

Cup of ice

42O water bath

Masking tape

1 LB/AMP plate

pGreen DNA

p20 volume
micropipette

37O incubator


1
clear
culture
tube

2 disposable
loops

p1000 volume
micropipette

10%
Clorox




Procedures:


Note: Unit of
measurement for the micropipette is microliters (ul). 1000ul = 1ml

1. Wipe lab table with 10%
Clorox

solution or detergent wipe before beginning work.

2. Set large micropipette (p1000

or a p200)
and with a sterile tip, transfer 250 ul of CaCl2 to clear
culture tube.

Note, if using p200 set device for 200 ul, and then repeat with setting at 50 ul.


3. Place tube on ice.

4. Go up to demo table and teacher will assist you in using a sterile disposable loop to transfer one or
two large bacterial colony into

the tube.

5. With a new tip, use the large
micropipette

to re
-
suspend the cells by pipetting up and down slowly.
Hold tube up to light to check if cell mass has been re
-
suspended.


6. Place the tube back on ice.

7. Using the small micropipette (p2
0) with a sterile tip, add 10 ul of the pGreen DNA plasmid directly
into the cell suspension. Tap the side of the tube with your finger to mix.

pGreen

4528 bp

Bioluminescence
Gene

(GFP)

Ampicillin
Resistance Gene

8. Place the tube back on ice and incubate for 5 to 10 minutes.

9. While the cells are incubating, use a
permanent marker to label the bottom edge of the LB plate
with your team name, period and LB. Label
the LB/AMP plate (has red stripe on side) the same way
but with LB/AMP. See diagrams below.












10.
CRITICAL STEP!

Carry the ice cup with cell suspension tube in it over to the 42O water bath.
Immerse the tube in the water bath for
exactly 90 seconds.



11. Immediately return the tube to ice for
at least 1 minute

for the cells to recover. Remove and hold
at room temperature for the rest of the lab.


12. Use a large micropipette (p1000

or p200
) with a sterile tip and add 250 ul
of LB broth to the tube.
Gently tap the tube with your finger to mix.


13. With a new tip
, transfer 100 ul of the cell suspension from the tube to the center of the LB plate.
Also transfer 100 ul of the cell suspension from the tube to the center of t
he LB/AMP plate.


14. Gently spread the suspension evenly across the LB plate using a clean sterile disposable loop. Go
back and forth while turning the plate in a clockwise motion.
BE CAREFUL NOT

to penetrate the
surface of the nutrient agar.


15
. Repeat the spreading process on the LB/AMP plate. You may use the same loop. Bring the loop
to the front of the classroom and insert in Erlenmeyer flash with bleach for disposal.


16. After 2 minutes tape both plates closed. Seal the plates with masking tape and place them
UPSIDE DOWN in the 37OC incubator for 24 hours.


Notes:





Petri dish with
nutrient agar

Label the under side

Name, Period

LB only plate

LB/AMP plate

-

Has Red stripe

Name,
Period

LB

LB/AMP


Name____________________________________________Period_____ Due Date________________




Genetic Engineering

Lab: Bacterial Transformation with pGreen



Experiment Data:


Draw your LB only and LB/AMP plate growth results.
Label plate the transformed bacteria.















LB only







LB/AMP


1. Which plate is the positive control? ______________________________________________.


2. Which plate is the experiment plate? ______________________________________________.


3. What is different in the experim
ental plate from the control plate? ______________________.


4. On which plate do non
-
transformed and transformed bacteria grow? ____________________


5. On the plate for question 4, why don’t you see both types of bacteria? ____________________

________
___________________________________________________________________________
___________________________________________________________________________________.


6.
TWO QUESTIONS:

On which plate do transformed bacteria grow well
and
why must they be
“trans
formed” to survive on this plate?
__________________________________________________________________________________

__________________________________________________________________________________

_______________________________________________________
___________________________

Conclusion:

What is the purpose of using a plasmid
with the bioluminescence gene
?

___________________________________________________________________________________
___________________________________________________________________________________
___________________________________________________________________________________

pGreen

4528 bp

Biol
uminescence
Gene

(GFP)

Ampicillin
Resistance Gene