pGLO Mutagenesis Session 2

Content objectives:

To understand how mutations can alter gene expression.

To understand the principles of bacterial isolation.
To understand how restriction enzymes function.

Scientific method objectives:

To perform data analysis.

To use computer simulation to design an experiment.

Laboratory skill objectives:

To demonstrate skill in aseptic technique and bacterial isolation.

Preparation and Assignments

Before lab:
NOTE: For each laboratory session you will be expected to have read and
understood the material for the days lab session and have taken notes on any
procedural videos before arriving in class.

Read this lab manual chapter

Complete the pGLO: Session 2 Pre-lab assignment, which
is due at the start of class

During class:
Review the pGLO: Session 2 Pre-lab assignment
Answer verbal questions posed by your Laboratory Instructor
Design your restriction digestion experiments using computer
simulation.
Start your pGLO: Session 2 Post-lab assignment.
After class:
Complete your pGLO: Session 2 Post-lab assignment- This
assignment is due at the start of pGLO: Session 3.

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Introduction
Isolating Mutants
In this lab, your goal is to isolate E.coli cells expressing the G- mutant
phenotype, i.e. those cells that do not glow in the presence of arabinose. In
the last session, you transformed competent E.coli cells with wild-type (Wt)
or mutant (M) pGLO plasmids and plated the transformed cells on selective
media. In this session you will look at these plates to find the G- mutant
phenotype. It is necessary that you isolate G- mutants alone and not E.coli
strains that may carry more than one mutation in the pGLO plasmid
(especially double mutants that could also have an insertion in the ampicillin
resistance gene).
Patching
There are many ways of isolating mutants. Patching is the technique of using
a sterile toothpick to lift a whole bacterial colony off an agar plate and
transferring it to the surface of a fresh agar plate. Patching can do 2 things
for you: verify the phenotype of mutants and give you enough DNA to work
with for subsequent analysis. In this session, you will use patching to 1)
verify the phenotype of your G- mutants and 2) produce enough cells so that
you have sufficient copies of your mutated pGLO plasmid DNA for restriction
digestion and sequencing analysis. In the next parts of the experiment, you
will use restriction digestion and sequencing analysis to determine where
exactly the EZ-Tn5 transposon has inserted into the pGLO plasmid.
Restriction Digestion
Remember that E.coli cells with the G- mutant phenotype contain a pGLO
plasmid with a transposon insertion in either the araC or GFP genes. You
would like to know which of these two genes has been mutated but you
cannot tell that by examining the phenotype alone. One way to determine
where the insertion has occurred is to first isolate the mutated pGLO plasmid
DNA from the E.coli cells you will patch today (you will do this next week in
pGLO: Session 3) and then use restriction digestion analysis to determine
whether the araC gene or GFP gene contains the EZ-Tn5 transposon insertion
(you will do this in pGLO: Session 4). It is important that you design an
appropriate restriction digestion experiment so that you will be able to
determine which of the two genes, araC or GFP, has been mutated. In
todays lab session you will perform a simulated digestion of the wild type
pGLO plasmid using computer software to determine which restriction
enzymes you should use to digest your mutant pGLO plasmid DNA so that
you separate the araC gene from the GFP gene.
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Restriction enzymes (REs), produced by microorganisms, are enzymes that

recognize a short specific base sequence in a DNA molecule and cleave the
DNA at that point. Microorganisms that produce REs can immediately digest
a foreign piece of DNA once it enters the cell if the DNA carries the base
sequence specific for those REs. This is a major defense mechanism that
many bacteria use for the destruction of invading viral DNA. Because this
foreign DNA is restricted from action in these bacteria (i.e. it can no longer
function within the host cell because it has been cut) the term "restriction
enzyme" or "restriction endonuclease" is used to describe this class of
enzymes.
You may ask yourself why the REs produced by a microorganism do not
digest its own DNA? The reason is that REs recognize only specific short
sequences of bases in the DNA molecule and either the host DNA does not
contain the these sequences or if such sequences exist, they are modified in
a way that the REs cannot recognize them. The most common type of
modification is the addition of a methyl group to one or more of the bases of
the recognition sequence. Methylation does not alter the coding capacity of
the DNA.
The discovery of REs and the mode of their action paved the way for
molecular cloning and genetic engineering. In 1970, Hamilton Smith was the
first to isolate an enzyme called HindII from Haemophilus influenza, which
could cut the DNA of phage T7 (a virus) into 40 specific fragments. When the
ends of the DNA fragments from the digested phage T7 DNA were
sequenced, it was found that all fragments had either a "C-A-Pu" or a "Py-TG" (Pu stands for purine and Py for pyrimidine). Smith concluded that HindII
acted at a very specific point on the DNA molecule as is shown below:

5'
-G-T-Py-Pu-A-C- 3'
| | | | | |
3'
-C-A-Pu-Py-T-G- 5'

In the above diagram, shows the site of action of the enzyme. Interestingly
these sequences also exist in the host Haemophilus influenzae from which
the HindII was isolated; however, the adenines are methylated and as such
HindII is not able to recognize the sequence in the host.
Another example of a common RE is EcoRI, which as the name indicates, is
produced by E. coli. The gene for this RE is on an R plasmid (a plasmid
conferring resistance to antibiotics). Its cleaving action is as follows:

5'

-G-A-A-T-T-C-
| | | | | |
3

3'

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3'

-C-T-T-A-A-G-

5'

This sequence exists in the virus SV40 and also in the E. coli itself, but again
in E. coli one of the adenine bases on each strand is methylated.
It is apparent from these two examples that no matter which strand is cut
with the RE, the sequence of the bases at the ends are identical if you turn
one fragment 180 over the other. (This is due to the palindromic--twofold
symmetrical--nature of the sequence of bases on which REs are active.)
Note that in HindII the point of action of the RE () on the two strands
coincides while for EcoRI, the points are 4 bases apart. Since the two strands
of the DNA stay together, HindII produces clean cuts called "blunt ends"
while EcoRI produces staggered cuts or 5'-overhanging ends called "sticky
ends". In both of the examples above, the recognition sequences are 6
bases long; however, many REs can recognize specific sequences of from 4
to over 20 bases. On the average, REs that act on fewer bases cut a DNA
molecule more times than those that act on longer base sequences. Finally,
REs are commonly named using 3-4 letters and a Roman numeral. The
letters designate the microorganism from which the enzyme was isolated
and the Roman letter indicates different enzymes from the same source.

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pGLO Mutagenesis: Session 2 Procedure

Overview
In this lab session, students will examine the results from the
experiment they conducted in pGLO: Session 1. This data will be
recorded in their lab notebooks and also used to answer the
questions in the pGLO: Session 2 post-lab assignment. Each
student pair will then patch G- mutant colonies onto selective
media. Once patching is complete, students will work in pairs to
design a restriction digestion experiment using computer
simulation software. Students will submit this information to their
Lab Instructor or TA before leaving lab so that we may order all
necessary REs the students would like to use in pGLO: Session 4.
Materials needed

Sterile blunt end toothpicks

L+Kan+Ara+Amp media plates (1 plate per pair of students)
UV lights
Protective eyeglasses
Sharpie pens in different colors for counting colonies
Laptops
NEB Catalog
RE Inventory List

Procedural Notes
Data Analysis of pGLO: Session 1 Experiment
1.

Examine the plates that you made in pGLO: Session 1. Document

the results from these plates, including counts of glowing and nonglowing colonies, in your lab notebook and in the pGLO: Session 2
post-lab assignment. Discuss whether or not these results make
sense. Make sure to share your data with the other groups at your
lab table.
When counting glowing colonies, please follow theses
directions:
-Put on protective eye goggles and turn on "long wave" UV lamp.
-This procedure works best if you have a dark background behind
the plates so turn off the lights in the classroom and/or move to a
dark place in the room.
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-Hold the lamp over the inverted plates and move it to make sure
you cover all parts of the plate.
-Do not forget to turn off the lamp after you are finished.
2.

Once all data has been collected, discard the wild-type pGLO
plates ONLY in the biohazard bins located at the front of the room.

3.

Keep the mutant pGLO plates. These are the plates you will use
for patching.

Isolation of G- Mutants via Patching

1.

Obtain a new L+Amp+Kan+Ara plate and label it (on the bottom

near the edge) with all necessary identifying information (Section #,
group name, media, pGLO 2).

2.

Wearing goggles and using the UV lamp, circle the G- mutant

colonies with a fine-point marking pen on the mutant pGLO plates
(be sure you DO NOT use the positive and negative control plates
from the mutant pGLO screen for this activity). These will be the
colonies you will patch in todays lab.

3.

Each pair of students will need a minimum of 2 unique G- colonies

for the next session, however it is strongly encouraged that you
patch as many G- colonies as possible from the experimental plates
(up to 20 mutants/pair). If you have very few G- colonies, you may
borrow some from other groups that have them in abundance.

4.

Watch the demo by your Lab Instructor or TA on how to patch Gcolonies onto your new L+Amp+Kan+Ara plate.
-To make patching easier, ask your Lab Instructor or TA for a grid
that you can place under the new L+Amp+Kan+Ara plate.
-Be sure to use a fresh toothpick for each G- colony and discard
the used toothpicks in the waste beaker on your lab bench.

5.

Invert the newly patched plate and incubate it at 37C for 40-48
hours. Your Lab Instructor or TA will store them for you in the lab
refrigerator until the next session.

6.

Record the procedure you used for isolating G- mutants via patching
in your lab notebook.

Restriction Digestion Simulation

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1. Working in pairs, open your browser and go to the NCBI site at

http://www.ncbi.nlm.nih.gov . In the search box, change the drop down
menu from "All Databases" to "Nucleotide" and in the lower box, type
the formal name of the pGLO plasmid, "pBAD-GFPuv", and then click on
"Search".
2. A new page will appear that contains information about the pGLO
sequence. Look through the data carefully and note the size of
the plasmid, the genes present, the location of each gene on the
plasmid and the sequence of bases.
3. Write down the GenBank number for the pBAD-GFPuv entry
here:
a. GenBank number: _____________________________
4. We will be purchasing your restriction enzymes from the
company New England Biolabs. This company provides an online
tool to run simulated restriction digestions. Open this website at
http://tools.neb.com/NEBcutter2/.
5. Enter the GenBank number for pBAD-GFPuv plasmid in the
appropriate box.
6. Towards the bottom left of the gray box select: This sequence is
CIRCULAR The select Submit on the right hand side of the
gray box.
7. A map of the wild-type pGLO plasmid with several (but not all)
restriction enzyme cut sites will appear once the simulated
digest is complete. Fill in the following information as it pertains
to this map:
a. What gene is represented at position a? ________________
b. What gene is represented at position b? ________________
c. What gene is represented at position c? ________________
8. You want to design a restriction digest experiment that will
separate the araC gene from the GFP gene. Looking at the map
of pGLO and the list of REs available for you to use in your
experiment (ask for this list from your Lab Instructor or TA),
select 2-3 enzymes that might be good for you to use in your
experiment. Note this map will show you several, but not all of
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the restriction enzymes that cut the pGLO plasmid. To see a full
list of enzymes that cut the pGLO plasmid select Custom
Digest at the bottom left-hand part of the page.
9. Once you have selected the restriction enzymes you want to use
in your experiment you can now perform a custom digest of the
pGLO plasmid using only these enzymes following the
instructions below.
10.
First, select the statement towards the top of the new page
that says Enzymes with compatible buffers. Check to see if the
enzymes you would like to use are grouped under a single buffer
type (either the CutSmart Buffer, NEB Buffer 1.1, NEB Buffer 2.1,
or NEB Buffer 3.1). If any of these enzymes are not found in the
same buffer system as the other enzymes you have selected,
you will need to select a different set of enzymes as you can only
add one buffer solution to your restriction digestion reaction.
11.
Once you have selected enzymes that are grouped under a
single buffer type, you will need to check if each enzyme has
100% activity in that buffer system. If any of the enzymes do not
have 100% activity you will need to select a different set of
enzymes as it is critical that the all the enzymes in the digestion
reaction work with 100% activity in the same buffer system.
12.
Once you have selected 2-3 enzymes with a 100% activity
in a single buffer system, click on the boxes next to the enzyme
names and select the green Digest button at the bottom of the
page.
13.
Examine this new pGLO map that shows only your selected
restriction enzymes cut sites and confirm that the enzymes you
have selected would separate the araC gene from the GFP gene.
14.
Towards the bottom left-hand side of the page that has
your new pGLO map you will find the selection View gel. Select
this button.
15.
This new page will show you the # of DNA fragments that would
be produced by this digest (upper right-hand side) and their lengths.
Note the # of fragments that would be produced and their lengths.
Based on this information answer the following questions: 1) Is there
more than 1 fragment for any given length? and 2) do any of the
fragments differ in length by less than 200bp? If the answer is yes to
either question you will need to redesign your experiment. Discuss
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why you would need to redesign in these cases with your Lab
Instructor or TA.
16.
Finally, you need to confirm that the enzymes you have
selected will not cut the EZ-Tn5 transposon DNA that has been
inserted into either of these genes. To do this first go to
http://www.epibio.com/tech-support/dna-sequences
17.
Select EZ-Tn5 <Kan-2> [1221bp] link. When the
sequence appears on the next page, select Text File at the top
of the page.
18.
When the text file of the sequence appears on the next
screen, copy the EZ-Tn5 sequence.
19.
In a new tab, open NEB Cutter at
http://tools.neb.com/NEBcutter2/ and paste the EZ-Tn5 sequence
into the white box. Select This sequence is LINEAR and and
then select Submit.
20.
This new map will show several (but not all) restriction
enzyme cut sites in the EZ-Tn5 DNA sequence. To see a full list
of enzymes that cut EZ-Tn5 select Custom Digest at the bottom
left-hand part of the page.
21.
Scroll through the list of enzymes. If any of the enzymes
you would like to use in your experiment appear in this list (and
thus cut the EZ-Tn5), you will need to redesign your restriction
digestion.
22.
Now that you have selected 2-3 enzymes that work with
100% activity in the same buffer system, produce DNA
fragments of unique lengths that are at least 200bp different in
size, and do not cut the EZ-Tn5 DNA, you should plan how you
will actually carry out the experiment in the lab. Using the NEB
Catalog, look up the information below for each enzyme that you
are proposing to use in your experiment. Based on these
answers, you may wish to redesign your restriction digestion
experiment to make it logistically feasible for pGLO: Session 4.
a. What is/are the incubation temperate(s) and lengths of
incubation for your enzymes?
b. Do any of the enzymes require heat deactivation? If so,
what is/are the incubation temperate(s) and lengths of
incubation for deactivation of your enzymes?
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c. Do any of your enzymes require the addition of BSA? Which

ones?
d. Draw a timeline for your experiment that includes the
order in which enzymes should be added to the reaction
(and if they require the addition of BSA), the temperatures
& lengths of incubation, and the temperatue and lengths of
incubation for deactivation.
e. How long will your experiment take to complete? (If the
answer is more than 2 hours, you will need to redesign
your experiment)
23.
Once you feel you have successfully designed your
restriction digestion experiment, answer the questions in the
pGLO: Session 2 post-lab assignment. Make sure to share this
information with your Lab Instructor or TA before you leave the
lab today. We will order the restriction enzymes you are
requesting and have them available for your use in pGLO:
Session 4.
Please note: Failure to share your restriction digestion
experiment with your Lab Instructor or TA before leaving today
may result in our inability to acquire the enzymes you need in
time for pGLO: Session 4. This situation will have a negative
impact on your grade.

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Name: ____Sebastian de Armas___

Section # __105_
pGLO: Session 2 Pre-lab Assignment
pGLO: Session 2

BIOL123-Fall 2014
Due at the start of

1. (1.5pts) One of the main objectives in pGLO: Session 2 is to isolate Gmutants from the plates you made in pGLO Mutagenesis: Session 1.
How do you propose to do this in todays lab?
We are looking for plates where the cells were resistant to both
kanamycin and ampicillin, and although being in the presence of
arabinose did not glow because of the transposon inserting itself into
the GFP DNA sequence
2. (0.5pts) What safety precautions do you need to take in todays lab
when working with the UV lamps?
Do not keep your body in the way of the UV rays for too much, wear
gloves and covering to protect from radiation. We will also need
protective eye goggles.
3. (1pt) What are restriction enzymes?
Restriction enzymes are enzymes that are able to locate and cut away
different (specific) pieces of DNA away from a DNA strand through
splicing. They are enzymes that recognize a short specific base
sequence in a DNA molecule and cleave the DNA at that point
4. (1pt) How do restriction enzymes aid in microbial survival?
Microorganisms that produce restriction enzymes can digest foreign
pieces of DNA once it enters the cell if the DNA contains the specific
sequencing that the restriction enzyme is looking for This is a huge
defense mechanism that many bacteria use for the destruction of
invading viral DNA (it essentially creates a specific immune system).
5. (1pt) List two mechanisms for how microbes protect themselves
against the activity of their own restriction enzymes?
Restriction enzymes only recognize short sequences in the DNA
molecule, and the case is usually that the host organism does not
contain those specific base pairings in its own genome or if they exist
the organisms has modified them enough so that the restriction
enzymes will not recognize them as foreign invaders. A common way
of doing this is the addition of a methyl group onto the DNA sequence.
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Name: __Sebastian de Armas_

Section # __105__

BIOL123-Fall 2014

pGLO: Session 2 Post-lab Assignment

of pGLO: Session 3
1.

Due at the start

(1pt) Fill in the following table with the data from your tables
experiments in pGLO: Session 1.

Table Avg # of
Table Avg # of Observations &
glowing cfus non-glowing cfusNotes
Wt-pGLO
xxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxx
-Positive control (10uL) 0
0
There were lots of
non glowing colonies
-Positive control (50uL) 0
0
There were tons of
non glowing colonies
-Negative control (10uL) 0
0
No colonies grew
-Negative control (50uL) 0
0
Only a few colonies
grew, they didn't
glow
-Experimental Plate (10uL)
746
0
All the colonies are
glowing
-Experimental Plate (50uL)
6804
0
All the colonies are
glowing
M-pGLO
xxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxx
-Positive control (10uL) 544
0
All the colonies are
glowing
-Positive control (50uL) 2000
0
All the colonies are
glowing
-Negative control (10uL) 0
0
No colonies grew,
they all died
-Negative control (50uL) 0
0
No colonies grew,
they all died
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-Experimental Plate (10uL)

42

10

-Experimental Plate (50uL)

64

18

2.

Some were glowing,

some were not
Some were glowing,
some were not

(1pt) Do the results make sense for the positive control plates? Why
or why not?
Yes the results make sense because on the wild type we see growth and not
glowing as the araC and GFP genes are left in tact and there is a lack of
arabinose and antibiotic meaning that the number of colonies will grow. The
results also make sense on the mutant type because in the presence of
arabinose and the insertion of a transposon into either the araC or GFP genes
we will see some glow and some not glow.

3.

(1pt) Do the results make sense for the negative control plates?
Why or why not?

The results do make sense for both the wild and mutant types as for the wild types
they are sensitive to kanamycin and therefore will die (producing zero of everything),
while the mutant types are resistant to kanamycin and ampicillin meaning that it will
survive in those conditions and continue to display the results of a transposon
insertion on glowing/not glowing.
4.

(1pt) Do the results make sense for the experimental plate for the
Wt pGLO experiment? Why or why not?
The results do make a lot of sense as the araC and GFP genes in the wild type
are left in tact, therefore in the presence of arabinose and ampicillin (which they
are resistant to) they will grow and glow (the GFP protein will be expressed,
leading to glowing) for essentially all the colonies.

5.

(1pt) Do the results make sense for the experimental plate for the
M pGLO experiment? Why or why not?
The results do make sense for the mutant pGLO experiment as some
will glow and some will not glow. The reason for this is because the
transposon may have inserted itself into either the GFP gene or the
araC gene, preventing the GFP gene from being expressed in the
presence of arabinose causing some to not glow.
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Restriction Digestion Experiment

6.

(0.5pts) Which enzymes would you like to use in your restriction

digestion experiment?
PciI and BsteII

7.

(0.5pts) Does digestion of the pGLO plasmid by these enzymes

separate the araC and GFP genes from one another?
Yes, the PciI cuts at 4748 bp and the BsteII cuts at 1077 bp

8.

(0.5pts) Do these enzymes work with 100% activity in the same

buffer system? Which buffer system will you be using in your
experiment?
No, the BsteII has a 50% activity at 37 degrees Celsius . We will be
using the NE buffer 3.1

9.

(0.5pts) Do these enzymes cut the EZ-Tn5 transposon?

They do not, yay!

10.

(0.5pts) Following restriction digestion, are the resulting DNA

fragments of different lengths? List the number of fragments
produced and their lengths here.

They are different lengths; there are 2 fragments:

Fragment 1: length of 3671 base pairs, cutting from 1078-4748 bp
Fragment 2: length of 1700 base pairs, cutting from 4749-1077 bp
11.

(0.5pts) Are any of the DNA fragments less than 200bp apart in
size? Why would it be an issue if your experiment produced
fragments less than 200bp apart in size?
No, and it would be an issue as when we would run the fragments
through the gel electrophoresis the fragments would be too close to
be able to tell the differences between them

12.

(0.5pts) Do any of your enzymes require the addition of BSA? If so,

which ones?

No

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13.

(1pt) Draw a timeline for your experiment that includes the order in
which enzymes should be added to the reaction and if they require
the addition of BSA, the temperatures & lengths for incubation, and
the temperatue and lengths for deactivation (if necessary).
Timeline:
At zero minutes we will add the PciI to the solution containing the
desired DNA fragments (add them to the DNA at 37 degrees celcius for
1 hour)
At 60 minutes we must raise the heat to 80 degrees celcius for 20
minutes (PcIs heat inactivation period)
At 80 minutes we must add the BsteII to the solution of DNA at 60
degrees celcius, so we have to lower the temperature from 80.
At 85 minutes we remove the entire solution from incubation as the
reaction is complete
24.

(0.5pts)How long will it take to complete your experiment?

85 minutes

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