Download pGLO LAB EDITED by ME

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University of Pittsburgh
“Glowing” Bacteria—Transformation of
E. coli with pGLOTM Plasmid
INTRODUCTION
Genetic transformation is a very useful tool to the molecular biologist because it
allows genes from one organism to be moved into another type of cell in order to
change the traits of that cell. Bacterial transformation is the process by which bacteria
take in and use a new piece of DNA. If the new DNA contains genes which the bacteria
did not already have, then the bacteria will be “transformed” and will exhibit new
traits. The molecular biologist begins by treating the bacteria cells with a solution of
calcium chloride in order to make cells that will more readily take up DNA—these are
competent cells. The positive charges of the chemical partially neutralizes the
negatively charged DNA and plasma membrane of the cell so that they will not repel
each other, allowing the DNA to pass through tiny pores in the cell membrane and into
the cell where it can be used to make proteins. We will be using a non-pathogenic strain
of E. coli bacteria that has already been treated with calcium chloride and is ready for
the transformation.
How do we know when transformation has occurred? Molecular biologists
select for bacteria that were successfully transformed by using the new traits, or
phenotype, of bacteria that contain the new DNA. A common selection method is
antibiotic resistance. If we begin with bacteria that will die when exposed to antibiotic,
and transform with DNA that contains a gene to give antibiotic resistance, then it is
likely that the cells that live when grown with the antibiotic have taken up the new
DNA. The DNA that we will be using for the transformation is a small circular piece of
DNA that contains a gene for ampicillin resistance--the pGLOTM plasmid. Ampicillin
(AMP) is an antibiotic similar to penicillin, and the ampicillin resistance gene produces
a protein called -lactamase that can destroy the ampicillin and allow the bacteria to
keep growing. The pGLOTM plasmid also contains a gene that will make the bacteria
glow when exposed to ultra-violet (UV) light by producing a protein called Green
Fluorescence Protein (GFP) when the bacteria is given a sugar called arabinose (Ara).
The arabinose is used as a “trigger” that switches the GLO gene on; therefore the
bacteria will glow only when they are grown with arabinose.
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We will begin the transformation by adding competent E. coli cells to two tubes;
the reaction tube (+DNA) to which we will add the pGLO plasmid, and the control tube
(-DNA) which will contain everything except the plasmid. After an incubation on ice,
the cells are heat-shocked at 42o C, cooled briefly on ice, supplemented with LB broth,
then incubated at 37 o C. After the last incubation, the cells from each tube will be plated
on three types of LB agar plates: LB only, LB/AMP, and LB/AMP/Ara. The agar plates
will be incubated for 24 hours at 37o C to allow the bacteria to grow. After the bacteria
have grown, the plates can be observed under white and UV light, and the results of the
transformation recorded.
Enjoy the project and keep your mind in full gear as you work!
EXPERIMENTAL PROCEDURES
Important Notes:
 Micropipettors are expensive and precisely calibrated instruments!
Ask for help before changing micropipettor setting!
 Never attempt to dial above or below volume limits printed on
micropipettor!
At the lab bench each group should have:
Ice bucket w/ice
2 15-ml push-cap culture tubes
Permanent lab marker
100-1000 l micropipettor and blue pipet tips
2-20 l micropipettor and yellow pipet tips
1 vial competent cells (keep on ice)
Purple 0.5 ml tube with TE buffer (TE)
Amber 0.5 ml tube with pGLO DNA (pGLO)
2 ml LB broth
1. Use permanent marker to label the two (2) sterile 15-ml push-cap culture
tubes as follows:
PD#-Group# --DNA = No DNA added / Control
PD#-Group#+DNA = pGLO transformation w/ plasmid added/ Experimental
Chill tubes by placing in ice bucket. Keep competent cells and culture tubes on ice as
much as possible!
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2.
Use the 100-1000 l micropipettor and sterile blue tip to add 100l of competent
cells to each 15-ml push-cap culture tube. Competent cells are delicate! Pipet
slowly to avoid damaging the cells! Pipet the cells directly into the bottom of the
tubes, so that a pool of cells forms at the bottom. Avoid splashing the cells up
onto the sides of the tube. Place tubes with cells back on ice. DO DNA + Tube
FIRST in case aliquot is short.
* NOTE: Even though it is always more accurate to chose the smallest
micropipettor that includes your desired volume in its’ range, this time you are
to use the 100-1000l and not the 20-200l to transfer 100l. The reason is
because the competent cells are so fragile that you want to use the bigger tip
(not the yellow) with the bigger micropipettor to avoid damage when being
transferred.
3.
Use the 2-20 l micropipettor with sterile yellow tip to add 10l of TE buffer
directly into cells in tube labeled --DNA. Close cap, tap tube with finger to mix.
Avoid making bubbles or splashing cell suspension up sides of tube. Return tube
to ice. This is the control tube.
4.
Use a fresh yellow tip to add 10 l of pGLO DNA directly into cells in tube
labeled +DNA. Close cap, tap tube with finger to mix, return tube to ice. This is
the transformation tube.
5.
Allow both tubes to incubate on ice for at least 15 minutes. (Cells could incubate
at this step for up to 45 minutes with no problems; 20 minutes seems to be the
optimal time.)
6.
While cells are incubating on ice, use permanent marker to label six pre-warmed
agar plates. Every group should have two of each of the following plates:
2 LB
black line
2 LB/AMP
black line + blue line
2 LB/AMP/Ara
black line + blue line + orange line
Divide plates into two sets of three plates, one of each type, and mark as
follows: In small print, label all plates around the circumference of the plate on
the bottom (agar-containing side) with your initials (be sure all group members
can ID from this), the date, and the agar type. One set of plates should also be
labeled with +DNA and the other set with –DNA. Each set of plates should have one
of each type: LB, LB/AMP, LB/AMP/Ara.
EX: PD#-Group# – 12/11/02 +DNA + (on 1 of each plate type for Experimental)
and
PD#-Group# – 12/11/02 -DNA+ (on 1 of each plate type for Control)
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7.
Following the 15-20 minute ice incubation, the cells in both tubes need to be heat
shocked.
Important Notes:
 Timing of the heat shock is very important! It is critical that cells
receive a sharp and distinct shock.
 Carry ice bucket with cells to water bath (or heat block) set at
42 o C and set timer for 45 seconds before starting heat shock step.
8.
Remove both tubes of cells from ice and immediately place into 42 o C water
bath (or heat block) and start timing 45 seconds. After 45 seconds, immediately
return both tubes to ice. Allow incubation on ice for at least one minute.
(Additional incubation on ice is acceptable, but it is better to proceed to the next
step.)
9.
Use a 100-1000 l micropipettor with a Large/blue tip to add 800 l of LB broth
to each tube. Use a fresh tip each time. Gently tap tubes with finger to mix.
* If you do not use a fresh tip, you could cross contaminate from Experimental
to Control and ruin the experiment!!!!!
10.
Allow cells to recover by incubating both tubes at 37oC (heat block, water bath,
or incubator) for at least 20 minutes. Additional time at 37oC is acceptable and
will lead to higher transformation efficiency. After 37oC incubation, cells will be
plated on agar plates that have been pre-warmed at 37oC. Read plating protocol
carefully before starting.
Important Notes:
 Plating cells requires the careful use of Bunsen burner and ethanol.
Extreme caution must be taken to prevent catching ethanol, hair,
clothing, or paper on fire!
 Always hold spreader downward to prevent ethanol drops from
falling onto hand or arm!
 If beaker of ethanol catches on fire, DO NOT TOUCH BEAKER—
IT WILL BECOME EXTREMELY HOT!! Call for help to cover
beaker and smother flame.
11.
a. Lift lid off of 1 of the three agar plates labeled –DNA; do not place lid down
on lab bench. Using a 100-1000 l micropipettor with a sterile large/blue tip,
add 250l of cell suspension from tube labeled --DNA onto the agar plate
marked --DNA. (The volume plated may be adjusted due to the length of
time at 37oC, in step 10. If the 37 o C incubation time was over 1.5 hours, then
only plate 50l of cells using a 20-200 l micropipettor with yellow tip.) Do
not allow suspension to sit on plate too long before spreading!
b. Remove spreader from ethanol and briefly pass through flame until ethanol
on spreader catches fire. While holding spreader downward, allow ethanol
to burn off away from flame.
c.
Cool spreader by gently rubbing it on surface of the agar away from cells.
After cooling, touch spreader to puddle of cells on agar and gently drag it
back and forth several times across surface of plate. Rotate plate a quarter
turn and continue spreading; repeat several times. Be careful not to gouge
agar. *Failure to COOL SPREADER could lead to Killing Bacteria!!
d. Replace plate lid. Return the cell spreader to ethanol without flaming. Repeat
step 11 with all agar plates, being careful to match tube and plate labels,
and using fresh sterile tip to transfer cells to each plate. * You have two
more –DNA plates and ALL 3 +DNA plates to do. Use the +DNA
cell suspension when plating the +DNA plates. SEE STEP 12 to
HELP!
12. Use matrix below as a checklist as --DNA and +DNA cells are spread on each
type of agar plate. Match labels on tubes with plate labels! It will be very difficult to
screen for transformants if you plate the cells on the wrong plate!
Check off each plate as you go!!!!!!!!!!
Plate Type
Plate Label
--DNA Cells
on –DNA plates
+DNA Cells
on +DNA plates
LB
black
____ 100l
____ 100l
LB/AMP
black/ blue
____ 100l
l
LB/AMP/Ara
black/ blue/
orange
____ 100l
____ 100l
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13.
Let plates sit for several minutes, agar-side down, to allow suspension to become
absorbed into agar. After a few minutes, tape set of six plates together, making
sure that the plates are all in the same orientation, i.e. agar-side is facing the
same direction.
14.
Place plates agar-side up in 37oC incubator, and incubate 12-24 hours.
15.
Take time for responsible cleanup:
a. Any disposable items that have come in contact with E. coli--culture plates
and tubes, micropipettor tips--should be placed into burn box, autoclave
bag, or bleach solution.
b. Discard liquid cultures and broth into liquid waste container.
c. Wipe down lab bench with 10% Lysol solution.
d. Wash hands before leaving lab.
16.
After plates have incubated for 12-24 hours and colonies appear, examine each
plate under white and UV light and record observations in the table. Bacterial
growth that completely covers the plate is called a lawn. If distinct colonies are
observed but are too numerous to be counted, it can be recorded as TNTC (too
numerous to count). If colonies are countable, record number.
-DNA
+DNA
Control
Experimental
Your Growth:
Your Growth:
LB
Glow? Yes/No
Glow? Yes/No
(4pts each)
LB/AMP
(4pts Each)
Correct? Yes/No
Correct? Yes/No
Explain What Growth/Glow
should be and WHY:
Explain What Growth/Glow
should be and WHY:
Your Growth:
Your Growth:
Glow? Yes/No
Glow? Yes/No
Correct? Yes/No
Correct? Yes/No
Explain What Growth/Glow
should be and WHY:
Explain What Growth/Glow
should be and WHY:
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LB/AMP/Ara
(4pts Each)
Your Growth:
Your Growth:
Glow? Yes/No
Glow? Yes/No
Correct? Yes/No
Correct? Yes/No
Explain What Growth/Glow
should be and WHY:
Explain What Growth/Glow
should be and WHY:
ADDITIONAL: If you did NOT get the correct growth, you must PROVIDE an
EXPLANATION on the back of this SHEET to EXPLAIN what your ERROR
could have been!! You will be graded on this too!!!
Name: __________________________ PD: _______
SCORE:
/ 25
QUESTIONS
UNCHECKED COPY TO BE ATTACHMENT 2 FOR EACH GROUP MEMBER IN LAB
WRITE-UP!
I WILL HAVE YOU MERGE GROUPS AND TURN IN A FINAL ? COPY TO BE GRADED
FOR ACCURACY!
1. What type of material is added to the E. coli bacteria in order to introduce new traits
in the bacteria? Include the new traits in your answer. (3pts)
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2. Would adding Green Fluorescent Protein to the cells give the same results as the
transformation? Think about the long term and if the offspring will be able to glow!!!
Explain in Detail what the difference is. (4pts)
3. Why are the E. coli bacteria treated with calcium chloride for the transformation?
Explain what it does and what we call the cells that have been treated with it. (3pts)
4. What is the purpose of the negative controls (the –DNA tube) in the experiment and
how did it differ from the experimental groups (the +DNA tube)? (3pts)
5. What allows some E. coli colonies to grow on LB/AMP plates after the
transformation? Explain what is going on based on the Central Dogma!! (3pts)
6. Why did the +DNA LB plate show a LAWN and the +DNA LB /AMP show TNTC.
To answer this, think about what occurred in some of the bacteria but not all during
the experiment!! (3pts)
7. Along with UV light, what else must be present to allow the transformed bacteria to
glow and why? (3pts)
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8. What is the advantage for an organism to be able to turn genes like the pGLO on and
off? (3pts)
QUESTIONS
1.
What type of material is added to the E. coli bacteria in order to introduce new
traits in the bacteria? Would adding Green Fluorescent Protein to the cells give
the same results?
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Plasmid DNA with the genes for green fluorescent protein and ampicillin
resistance ( lactamase) is added to the bacteria. Adding GFP directly to the
bacteria may allow it to glow temporarily, but the offspring would not be able
to produce the GFP without the gene, and therefore would not glow.
2.
Why are the E. coli bacteria treated with calcium chloride for the transformation?
The E. coli bacteria are treated with calcium chloride in order to help
neutralize the negative charges on the cell membrane and the DNA so that the
DNA can more readily enter into the bacterial cells.
3.
What is the purpose of the negative control (the –DNA tube) in the experiment?
The negative control allows us to better evaluate the results of the experiment.
The cells did not grow with ampicillin before the transformation and did grow
with ampicillin after the addition of the plasmid DNA, therefore the DNA
must have affected the ability of the bacteria to survive in the presence of the
antibiotic.
4.
Why do E. coli colonies grow on LB/AMP plates after the transformation? If a
sample of transformed bacteria from the LB plate is grown on an LB/AMP plate,
will they grow?
The bacteria that have been transformed with the plasmid containing the
ampicillin resistance gene are able to produce -lactamase, a protein that
destroys the ampicillin. A sample of transformed bacteria from the LB plate
may or may not grow on the LB/AMP plate, depending on whether or not it
has taken up the plasmid DNA.
5.
What factors must be present to allow the transformed bacteria to glow?
The arabinose sugar as well as ultraviolet light must be present in order for the
bacteria to glow. The presence of arabinose allows the gene for the GFP to be
turned on, and the protein produced. The GFP uses the energy from the
ultraviolet light to emit a longer wavelength of light that is visible.
6.
What is the advantage for an organism to be able to turn genes on and off?
Producing proteins requires a great deal of energy from the cell, therefore it is
more efficient to produce only the proteins which are necessary for the
growing conditions of the cells.
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NAMES: ________________________________________________________________
(If Merging Groups – Put Group ID with Members from Each Group PLUS – If Growth is
Different between Groups, Please Indicate This Under Your Growth AND Explain at end)
-DNA
Control
Your Growth:
+DNA
Experimental
Your Growth:
Glow? Yes/No
Glow? Yes/No
Correct? Yes/No
Correct? Yes/No
Explain What Growth/Glow
should be and WHY:
Explain What Growth/Glow
should be and WHY:
LB
(4pts each)
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Your Growth:
Your Growth:
Glow? Yes/No
Glow? Yes/No
Correct? Yes/No
Correct? Yes/No
Explain What Growth/Glow
should be and WHY:
Explain What Growth/Glow
should be and WHY:
Your Growth:
Your Growth:
Glow? Yes/No
Glow? Yes/No
Correct? Yes/No
Correct? Yes/No
Explain What Growth/Glow
should be and WHY:
Explain What Growth/Glow
should be and WHY:
LB/AMP
(4pts Each)
LB/AMP/Ara
(4pts Each)
ADDITIONAL: If you did NOT get the correct growth, you must PROVIDE an
EXPLANATION below to EXPLAIN what your ERROR could have been!! You
will be graded on this too!!! If a Merged Group, this is only for the Group that
did NOT get accurate Growth – Denote Group #/ID.
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