Genetic Engineering
Lab: Bacterial Transformation with pGreen
Ampicillin
Resistance Gene
pGreen
4528 bp
Bioluminescence
Gene (GFP)
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 transformed 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 the 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 genetic. 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, and high
schools.
Purpose: To genetically transform the E. coli MM294 bacterium by inserted the pGreen plasmid.
Materials: Groups of 4 students
Starter colony
CaCl2 solution
plate
1 LB only plate
LB broth
1 LB/AMP plate
pGreen DNA
1 clear culture
tube
2 disposable
loops
Permanent
marker
Cup of ice
p20 volume
micropipette
p1000 volume
micropipette
Used tips/tube
container
42O water bath
37O incubator
Micro centrifuge
(optional)
Masking tape
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 (p20) 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.
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.
LB only plate
Name, Period
LB/AMP plate - Has Red stripe
Name, Period
Petri dish with
nutrient agar
Label the under side
LB
LB/AMP
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 the 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:
Name____________________________________________Period_____ Due Date________________
Genetic Engineering
Lab: Bacterial Transformation with pGreen
Ampicillin
Resistance Gene
pGreen
4528 bp
Bioluminescence
Gene (GFP)
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 experimental 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
“transformed” to survive on this plate?
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
Conclusion: What is the purpose of using a plasmid with the bioluminescence gene?
___________________________________________________________________________________
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