Transformation of Escherichia coli with pGAL

Background Info, Examples, Questions, etc.

Experiment Objective:
The objective of this experiment module is to develop an understanding of the biologic
process of bacterial transformation by plasmid DNA. This experiment introduces an
opportunity to observe an acquired phenotypic trait of the transformed bacterial cells.
Rev. 2/4/13

Transformation occuring at an adhesion zone of competent Escherichia coli

The Experiment:
Many different plasmids serve as useful tools in molecular biology. One example is the
pGAL plasmid which contains 6751 base pairs and has been cleverly modified by genetic
engineering. In the cell, it does not integrate into the bacterial chromosome, but replicates
autonomously.

Map of pGAL
In this experiment, students will transform host bacterial cells with a plasmid DNA. The
transformants exhibit a blue color and acquire antibiotic resistance, due to the incorporation
and expression of b-galactosidase and ampicillin resistance genes respectively. The number
of transformants will be counted and the transformation efficiency will be determined.
The plasmid pGAL contains the E. coli gene which codes for the enzyme -galactosidase.
Since the host E. coli does not contain a -galactosidase gene, only cells transformed by
pGAL will produce functional -galactosidase. In the presence of artificial galactosides
e.g. 5-Bromo-4 Chloro 3-indolyl--D-galactoside (X-Gal), pGAL-containing colonies
appear blue when colorless X-Gal is cleaved into a colored product.

Enzymatic cleavage of lactose by -galactosidase

Transformation by pGAL is also demonstrated by resistance to ampicillin. E. coli host cells
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used in this experiment are sensitive to ampicillin. pGAL contains the ampr gene that
encodes for the enzyme -lactamase which diffuses into the surrounding medium and
inactivates ampicillin.
- With time, small white colonies may appear around a large blue colony. Cells in those
small "satellite" or "feeder" colonies are not resistant to ampicillin and have not been
transformed with the pGAL plasmid. They are growing in a region of agar where the lactamase from the pGAL-transformants has diffused and inactivated the antibiotic
ampicillin. The number of satellite colonies increases if the concentration of ampicillin is
low or if the plates have incubated for extended times.

"Satellite" colonies about colonies of pGAL-transformed E. coli

Too much was TOO MUCH! Plates inoculated with too many transformants

Just Right!

Experiment Results and Analysis - Address and record the following in your laboratory
notebook or on a separate worksheet:
Before starting the Experiment:
Write a hypothesis that reflects the experiment.
Predict experimental outcomes.
During the Experiment - Data Collection - Observe the results you obtained on your
transformation and control plates:
Record (draw) your observations, or photograph what you observe. For each of the
plates, record the following:
How much bacterial growth do you observe? Determine a count. (A convenient
method to keep track of counted colonies is to mark the colony with a lab marking pen on
the outside of the plate. )
What color are the bacteria?
Why do different members of your class have different transformation efficiency
values?
If you did not get any results, what factors could be attributed to this fact?
Following the Experiment:
Determination of Transformation Efficiency - Transformation efficiency is a
quantitative determination of how many cells were transformed per 1 g of plasmid DNA.
In essence, it is an indicator of how well the transformation experiment worked. You will
calculate the transformation efficiency from the data you collect from your experiment.
(Calculation of transformation efficiency is explained below)
Formulate an explanation from the results.
Determine what could be changed in the experiment if the experiment were repeated.
Write a hypothesis that would reflect this change.
Experiment Results and Analysis
Answer these questions BEFORE analyzing your results.
1. On which plate(s) would you find only genetically transformed bacterial cells? Explain.
2. What is the purpose of the control plates? Explain the difference between each and why
it is necessary to run each.
3. Why would one compare plates AMP/X-GAL - PLASMID and AMP/X-GAL
+PLASMID?
Answer these questions AFTER analyzing your results.
1. Did you observe any satellite colonies? Why are the satellite,feeder colonies white?
2. Why did the competent cells which did not receive DNA (control) fail to grow on the
plates containing ampicillin?
3. Why are there so many cells growing on the X-GAL plate? What color are they?
4. What evidence do you have that transformation was successful?
5. What are some reasons why transformation may be unsuccessful?
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Example
Photo Identification Label: TUES 8/4/09 -

Followed Directions Properly labeled plates & tape, protocol steps 6 &11?
Type of Media

Treatment

Your Initials

Date

Initials and the date on the tape

Discuss your results. Was your experiment successful? In contrast to the other students
who performed the same experiment, how successful was your attempt?
?... Answer will vary for individual students
Review the notes that you should have taken as you performed the experiment. If your
experiment was unsuccessful, or marginally successful, offer plausible explanations for
your results.
?... Answer will vary for individual students

Another plasmid is also available in kits designed to teach bacterial transformation.

Transformation with pGLO produces colonies that glow under UV radiation, rather than the
blue colony phenotype produced by pGAL.
In the experimental results below, assume that the pGLO was used at a concentration of
0.1 g/L. Calculate the efficiency for each of the four experiments below. Explain how
you achieved your answer, i.e. show the math.
Experiment #1
Given: 0.1 g/l plasmid, 250 l CaCl2, 250 l LB broth, 10 l of plasmid added to culture, 100
l of solution from the reaction tube spread on agar plate
Results: 44 colonies on the + plasmid ampicillin plate
a. Determine mass of the plasmid added to the + plasmid transformation reaction tube.
0.1 g/l x 10 l = 1.0 g plasmid
b. Determine the concentration of plasmid in the reaction tube
1.0 g (a)
_______________ = 0.00196 g plasmid/l
(250 l LB broth + 10l Plasmid + 250l CaCl2)
c. Determine the mass of plasmid in the solution added to the agar plate
0.00196 g plasmid/l x 100 l = 0.196 g plasmid
d. Determine the number of colonies on the + plasmid LB/Amp plate per g of plasmid
44 transformed colonies = 225 colonies per g of plasmid
0.196 g plasmid

Experiment #2 ?

Experiment #3 ?

Experiment #4 ?

Assuming that the aliquot of pGLO that you used had a concentration of 0.1 g/L,
calculate the efficiency of your experiment. Explain how you achieved your answer, i.e.
show the math.
?... Answer will vary for individual students
What phenomenon can be observed on this plate?
Offer at least one plausible explanation for such a
result.
Satellite colonies - Ampicillin is not bactericidal
antibiotic after extended incubation, the E. coli that
were not transformed will grow into colonies. The
ampicillin in the plate was eventually destroyed by the
beta-lactamase that was produced by the successful
transformants (by their expression of the bla gene on
pGLO)

gmuller@framingham.edu

Transformation of Escherichia coli with Plasmid DNA

(pGAL)
Laboratory Protocol
Rev. 2/4/13

Transformation occuring at an adhesion zone of competent Escherichia coli

Each Lab Station of 2 Students Will Need:

Marker pen

Optional

Sterile disposable loops

Foam microtube holder/float

Micropipettes (p20 and p200) and Pipette tips

Transfer pipettes

vs.

Biohazard disposal container

Media Plates

Transfer pipettes

1 LB/X-gal agar plate (w/ no stripe)

2 LB/X-gal/Amp agar plates (striped)
Microcentrifuge tube containing 25 L pGAL plasmid (P)

on ice

Microcentrifuge tube containing 25 L CONTROL BUFFER (C)

on ice

Microcentrifuge tube containing 700 L competent E. coli cells (Ec)

on ice

Microcentrifuge tube containing 1500 L LB (recovery) broth (RB)

The Lab Will Also Need:
Ice-water bath(s)

at room temp

Preheated 42C water bath

37C incubator

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1. Label Tubes
a. Label the C (for CONTROL BUFFER)
microcentrifuge tube on its cap w/ your initials
b. Label the P (for pGAL DNA) tube
w/ your initials
c. Place both tubes back on ice

2. The Negative Control

a. Add 0.25 mL (250 L) of E. coli cells (Ec) into
the CONTROL BUFFER (C) tube
b. Mix by tapping
c. Place the tube back on ice
3. The Experimental Transformation
a. Add 0.25 mL (250 L) E. coli cells (Ec) into
the pGAL DNA (P) tube
b. Mix by tapping
c. Place the tube back on ice
e. Discard the used Ec tube into the biohazard
disposal container

4. Incubate on Ice
Incubate both tubes on ice for a minimum of
10-15 minutes (for up to 45 minutes)

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5 & 6. Heat Shock the Bacteria*

- At the end of the 10-15 minute incubation on
ice:
a. Bring your ice bath with the CONTROL (C)
tube and the pGAL (P) tube to the 42C water
bath
b. Transfer both tubes into the water bath for
exactly 90 seconds
c. After the 90 second incubation, immediately
place both tubes back on ice for a 2 minutes
* For best results, the transfer from the ice to the 42C

ICE 420C/90 sec ICE

water bath and back to the ice must be rapid

7 & 8. Add Recovery LB Broth

- After 2 minutes (step 5c, above), remove the
tubes from ice, then
a. Add 750L of LB broth into
the CONTROL (C) tube and close it
b. Add 750L of LB broth into
the pGAL (P) tube and close it
c. Mix both tubes by inversion

STOP POINT - After you have added the broth to the two tubes, you can leave the tubes at room
temperature overnight then refrigerate until you are ready to use them
- Tubes may remain in refrigerator for a week or more
- Let tubes warm up at room temperature for a half hour before plating

9. Recovery
Incubate the tubes at at 37oC for 30 minutes
(or at room temperature for >60 minutes)

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10. Label Plates (during the step 9 Recovery period)

While the tubes are incubating, label three plates
on their bottoms (not on their lids)
Label each plate on its bottom (agar side):
- Type of Media: X-gal (w/ no stripe)*
or LB/X-gal/Amp (striped)*
- Treatment : C (i.e. CONTROL) or P (i.e pGAL)*
- Your Name/Initials
- Todays Date
* i.e. Label the three plates as:
- X-gal C
- X-gal/Amp C
- X-gal/Amp P

11 - 16. Inoculate the Agar Plates with

Bacteria

Use a loop to spread the inocula

Using a micropipette and a fresh tip each time,

transfer bacteria from tubes to plates:
a. Transfer 250 L from the CONTROL (C) tube
to the LB/X-gal/CONTROL plate
b. Transfer 250 L from the CONTROL (C) tube
to the
LB/X-gal/Amp/CONTROL plate
Or simply rock the plate to spread
c. Transfer 250 L of the transformation from the

the inocula

pGAL (P) tube to the LB/X-gal/Amp /pGAL

plate

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17 - 22. Incubate the Plates to Allow Bacterial

Growth
a. Tape your three plates together & Write your
name(s) and todays date on the tape
b. Wait 15-20 minutes before turning plates
upside down
c. Incubate plates for 19-24 hours at 37C

23 - 24. Observe and Analyze

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When you are finished the lab exercise, wash your hands with soap and water!
gmuller@framingham.edu

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Transformation of Escherichia coli with pGAL

Teacher Preparation
The Day the Kit Is Received: Unpack & Store Components in
Frig/Freezer
Rev. 2/4/13

Preparation of Competent E. coli Cells:

The Day Before the Lab:

Reconstitute E. coli
a. Add 6 mL of Cell Reconstitution Medium (Component F) to the vial of lyophilized E.
coli cells (stored in the frig)
b. Replace stopper, mix by inverting until the freeze dried plug is dissolved, then shake
vigorously
c. Incubate at 34-37C for 16-24 hr (overnight, optimally for 19 hr)

The Day of the Lab:

Induction of Competency
a. Completely thaw the competency induction solvent (Component G) and store on
ice (if there is a white precipitate, warm if 37C water bath, then place on ice)
b. Mix by inversion to resuspend the reconstituted E. coli cells (above) and place on
ice for 10 min
c. Add 3 mL of cold competency induction solvent to the reconstituted E. coli cells
(competency induction solvent will be viscous, none should remain in the pipette)

d. Mix by inversion to form a uniform suspension

e. Incubate cells on ice for a minimum of 30 min (for up to 3 hr)

Day of the Lab (before students arrive):

Preparation of Aliquots of E. coli Cells
a. Mix cells by inversion to form a uniform suspension
b. Aliquit 0.7 mL (700 L) of cells into ice-cold microcentrifuge tubes
c. Cap, label them as Ec and immediately place tubes back on ice (can hold them on
ice for up to 3 hr)
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d. Remember to Prepare the 42oC water bath and ice-water buckets for class

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Preparation of Agar Plates:

1-2 Days before the Lab:

For optimal results, prepare plates two days prior to plating and set aside the plates inverted at room
temperature
If they are poured more than two days before use, they should be stored inverted in the refrigerator
when needed, remove the plates from the refrigerator and store inverted for two days at room
temperature before use
vs. If plates will be used within two days, store in a sealable plastic bag so the plates will not dry out
- store at room temperature, inverted)
vs. Cure for 2 days at room temperature, then bag and store in the refrigerator

Melt the ReadyPour Medium

a. Equilibrate a water bath at 60C
b. Loosen, but do not remove, the cap on the ReadyPour medium bottle

(to allow for the

venting of steam during heating)

c. Squeeze and vigorously shake the plastic bottle to break up the solid agar into
chunks
d. Heat the bottle of ReadyPour medium by one of the methods below (when completely
melted, the amber-colored solution should appear free of small particles)

- Microwave method:
Heat the bottle on High for two 30 second intervals
Using a hot glove, swirl and heat on High for an additional 25 seconds, or
until all the medium is dissolved
Using a hot glove, occasionally swirl to expedite melting
- Hot plate method:
Place the bottle in a beaker partially filled with water.
Heat the beaker to boiling over a hot plate or burner.
Using a hot glove, occasionally swirl to expedite melting.
e. Allow the melted medium to cool to 60C, it will be warm to the touch, not burning
hot (placing the bottle in a 60C water bath will allow the agar to cool, while preventing it from prematurely
solidifying)

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Preparation of Agar Plates (continued):

Pour the Plates

- Add reagents to medium which has been cooled to60C - hot medium will cause
reagents such as ampicillin to rapidly decompose
a. Open both sleeves of 20 plates; make 4 stacks of 10 plates
b. Thaw and add all of the X-Gal solution (component E) to the medium; Recap bottle
and swirl to mix
c. Use a fresh 10 mL pipet and a pipet pump to pour 10 LB/X-Gal control plates, 5
mL each (save the pipet for step e)
d. Add the ampicillin powder (component D) to the remaining medium; recap the
bottle and swirl to completely dissolve the ampicillin
e. Use the 10 mL pipet from step c and a pipet pump to pour 20 LB/X-Gal/Amp
plates, 5 mL each
f. Use a lab marker pen "stripe" the sides of the 20 LB/X-Gal/Amp plates (to provide an
easy method of differentiating between plates with ampicillin vs. plates without ampicillin)

g. Allow the agar to cool and resolidify

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Preparation of Aliquots of Buffers, pDNA, etc.:

Day of the Lab (before students arrive) vs. 1-2 Days before the Lab:
Preparation of Aliquots of Control Buffer and pDNA
a. Thaw the tubes of supercoiled pGAL DNA (component B) and control buffer
(component C) on ice (before dispensing, tap the tubes to knock all of the reagents to bottoms of
the tubes)

b. Using an micropipetter, dispense 25 L of the pGAL DNA into each of 10

microcentrifuge tubes; cap the tubes, label them as P (for pGAL DNA), and place
them on ice
- Rock the petri plate back and forth to obtain full coverage
- Pipet carefully to avoid forming bubbles - if the molten medium contains bubbles, they can be
removed by passing a flame across the surface of the medium

c. Using an FRESH micropipet tip, dispense 25 L of the control buffer into each of 10
microcentrifuge tubes; cap the tubes, label them as C (for Control Buffer), and place
them on ice
- The aliquots of pGAL DNA and control buffer may be prepared in advance,
refrozen and thawed in ice the day of the lab
Preparation of Aliquots of Recovery Broth
a. Dispense 1.5 mL Recovery Broth into 10 sterile tubes labeled "RB"
b. Cap the tubes and refrigerate if not to be used immediately
- Optionally,set up a classroom pipeting station

gmuller@framingham.edu

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10 Agar plates with X-GAL

no stripe plates: 5 ml each - ReadyPour medium without ampicillin
20 Agar plates with X-GAL and AMP
striped plates: 5 ml each - ReadyPour medium with ampicillin

Summary of Reagent Preparations

Dispense
10 plates Agar plates with
X-GAL (No Stripe) 5 ml
20 plates Agar plates with AMP
and X-GAL (Striped) 5 ml
10 tubes pGAL DNA 25 l on ice
10 tubes Control Buffer 25 l on ice
10 tubes Cells 0.7 ml on ice
Each Group Requires:
1 tube pGAL DNA 25 l on ice
1 tube Cells 0.7 ml on ice
1 tube Control Buffer 25 l on ice
1 agar plate - no stripe
2 agar plates - striped
4 sterile 1 ml pipets
2 sterile inoculating loops
1 tube Recovery broth (optional) 1.5 ml
2 micropipet tips (if using automatic micropipets)

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