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Bryan Fong
MIC 155L
Experiment 4
Isolation and characterization of carbohydrate utilization mutants in E. coli
Abstract
E. coli was mutagenized by a transposon and checked to see if we got any
mutations of interest. Our mutagenized cells were plated on LB/ Kan plates to verify if
our transposon has incorporated itself into the competent cells’ DNA because it has a
marker gene. From the replica plating of these cells on MacAra and MacLac, MacMal
plates to screen for mutations, the colonies were mostly pink indicating that the cells
could utilize the sugars. We did get a few possibly white colonies from the replica
plating, but when purified and screened again onto the respective MacConkey agar plates,
there were no sign of mutants because the colonies were all red. Some uncertainties
arose because the pure liquid cultures of the mutagenized cells did not grow in LB/Kan.
Also, we had problems isolating the DNA of the mutagenized cells because there seemed
to be little available.
Introduction
In this experiment, we wanted to induce mutations in E. coli using mariner
transposon by electroporation and see if it effects carbohydrate utilization. We will be
using the pFD1 mariner minitransposon to mutagenize E. coli. The transposon will
incorporate itself into the DNA of the bacteria. Before the transposon becomes inserted
to the bacteria’s DNA, the transposon must enter through the cell membrane. To do this,
the cells were electroporated so that there are holes in the cell membrane exposing the E.
coli’s DNA. The integration of the transposon into the E. coli is random. It can go
anywhere in the bacteria’s DNA. This means that the transposon can mutate any gene on
the bacteria’s chromosome, and by chance, can mutate a gene that affects carbohydrate
metabolism. The transposon used contains a kanamycin resistance gene, which acts as a
selectable marker to determine if the transposon is incorporated into the bacteria’s DNA.
Only when the transposon is integrated into the DNA will it express the Kanr gene
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because it the origin of replication requires pi protein, which is not present in our
bacterial strain.
A screen is done to determine where the transposon is incorporates into the
bacteria’s DNA. We can look for specific mutants to see if the transposon has disrupted
the genes. Bacteria cells from transposition can be screened on MacConkey agar plates
to see is they can utilize certain sugars. If the cells are mutagenized by the transposon,
then they will not be able to utilize the sugar and will be represented by a white or pink
colony. If we did find a mutant that cannot utilize a particular sugar, then our transposon
could be incorporated in this gene. From this, we can analyze the DNA sequence of the
bacteria and determine how the gene functions.
Once in the bacteria’s DNA, the transposon can be isolated with restriction
enzymes. The DNA fragment contains both the transposon and other gene sequence
surrounding the transposon. If the mutagenesis was inserted into a gene, then the regions
of DNA surrounding the transposon are parts of the gene it has disrupted. The pFD1
mariner transposon is special because it contains an origin of replication making it a
replicating plasmid. Once it is cut out by restriction enzymes, it can ligate itself back to a
plasmid. The plasmid with the transposon and gene can be taken in by another bacteria
cell that is a pir+ strain, which allows the replicating plasmid to survive without being
integrated into the bacteria’s DNA and cloned to make more copies. When we have
enough copies of the plasmid, the DNA can be sequenced containing the transposon and
the gene of interest. After sequencing, a BLAST search can be done to determine
location of the transposon and to identify the gene of interest that the transposon
surrounds.
Methods
Day 1
An aliquot of 50 l of “electrocompetent” E. coli cells were taken and put into an
Ependorf tube (epi 1) that was then placed on ice. Then 0.5 ml of LB was added to a 2
test tubes (labeled tube 1). A control tube (labeled control) is used containing 50 l of E.
coli, 0.5 ml LB, and 5 l of water. 2 l of pFD DNA is added to epi 1 and placed under
ice. A sterile electroporation cuvette was removed and placed under ice until the process
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was ready to start. The contents of epi 1 were added to the sterile electroporation cuvette
and placed in the electroporator at the settings:
Capacitance
25 F
Voltage
1.5kV
Resistance
200
After electroporation, the contents in the cuvette were removed and placed into tube 1
contain the 0.5 ml of LB. Both tubes were incubated at 37 C on the roller drums. After
one hour, 100 l was plated from tube 1 onto 5 LB/Kan plates. 100 l of the control was
plated on a series LB agar plates to get a viable cell count. The plates were incubated at
37 C for 24 hours.
Day 2
After incubation, replica plating was done on all 5 LB/Kan plates with
MacConkey lactose, maltose, and arabinose. Once this was done, the plates were
incubated at 37 C for 24 hours.
Day 3
From the original LB/Kan plates, 3 ml of M9 buffer is added to each these plates.
After 10 minutes, 1.4 ml of the suspension in each plate was taken out and put into vials
for freezer stocks. 0.8 ml of glycerol is added to the vials and the tubes were inverted.
Once finished, the vials are placed in the 80 C freezer.
From the replica plating, the mutants should be scored by the color of the
colonies. Colony purification onto LB/Kan and MacConkey (depending on the results)
were done to test what types of mutations we got.
Day 4
Overnight cultures were made from our LB/Kanr cells in 3 ml of LB/ Kan (0.2%
Kan) and placed in the roller drums for DNA isolation.
Next, the DNA was isolated to quantify the amount of DNA present. 1 ml of the
overnight was transferred to a 1.5 ml microfuge tube. The cells were in the microfuge for
1 minute. After the minute was over, the supernatant was removed and the resuspended
in 100 l of GTE and placed in ice for 5 minutes. 200 l of SDS/NaOH was added to
tube, inverted, and incubated at room temperature for 5 minutes. After 5 minutes, 150 l
of ice-cold KOAc was added to the tube, inverted, and placed in ice for 10 minutes. The
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tube was then spun for 15 minutes. The supernatant was transferred to a new 1.5 ml
microfuge tube and 400 l of ice-cold isopropanol is added and inverted. The
supernatant is then spun for 10 minutes. Once the microfuge has stopped, the supernatant
is removed and 200 l is added to the tube, gently inverted, and spun again for 10
minutes. Finally, the supernatant is removed and the pellet is air dried for 5 minutes.
After drying, 100 l of TE is added to the tube ready for DNA amount measuring.
Using a quartz cuvette, our DNA sample was diluted 1:200 in a final volume of 1
l. The optical density (OD) was then measured at wavelengths 260 and 280.
Change in Protocol (See Chromosomal DNA Mini prep)
Basically, this new protocol measures the amount of DNA from the cells on
LB/Kanr plates and the DNA is digeted and ran of a gel electrophoresis to see if we can
view the bacteria’s DNA. We did not get a lot of DNA so we couldn’t continue on with
the transformation part of the experiment.
Results
Amount of DNA isolated from transposon mutagenesis
Original protocol
Modified protocol
Amount of DNA
Amount of DNA
OD260 = 0.032
OD280 = 0.018
OD260 = 0.069
OD280 = 0.057
OD260/OD280 = 1.8027
OD260/OD280 = 1.2000
[DNA] = 320 mg/ml
[DNA] = 690 mg/ml
Electroporation constant = 4.8 sec
Transposon Mutagenesis
Plate #
1
Kanr
(colonies)
1500
Lac-
2
3
LB/Kan
4
1700
800
1200
MacConkey Lactose
0
0
0
0
Total # of cells = 4.28 x 10^7 cells/ml
5
1000
0
There were a few tiny white colonies on the MacLac plates that could be Lac- mutants.
These colonies were purified on LB agar and screen again on MacLac plates. The new
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MacLac plates showed all red colonies. On MacAra MacMal, and MacLac were mostly
red with cloudy dispersal around the colonies from the MacConkey agar.
Discussion
We did not get the results that we expected. However, we got Kanr cells because
there was growth of E. coli on the LB/ Kan agar plates. This means for the most part that
the transposition was a success. From the replica plating onto the MacAra agar plates,
the colonies were red indicating that the bacteria that we used can utilize the sugar
Arabinose. We were told that the strain of bacteria we using were already Mac-, and this
was verified by white colonies on the MacMal agar plates. However on the MacLac agar
plates, most of them were red with a few possibly white exceptions. The potential Laccolonies were purified on LB and then tested on back onto MacLac- agar plates. It shows
that the possible Lac- colonies could utilize the sugar lactose because the new MacLac
plates made all had red colonies. We did get transposition in our E. coli, we just did not
get mutants of interest. The transposition is a random event and could happen anywhere
on the bacteria’s DNA. If there was transposition of a mutant of interest, there may have
not been enough time (phenotypic lag) to express the mutated phenotype. In the future,
we could do a few things to improve the number of mutants of interest. We could use a
transposon that is specific gene that effects metabolism, increase the amount of
transposon used during electroporation, or increase the concentration of cells used during
electroporation. With an increase the concentration, the probability of getting a mutant of
interest is higher.
From determining the amount of DNA in our mutagenized cells from the original
protocol we got a OD260/OD280 ratio of 1.807. This means that we got pure DNA or
RNA. When the DNA undergone electrophoresis, there seem to be no DNA, only RNA.
rRna from the ribosomes are stable and were the only thing that was in the gel according
the ladder DNA that we used. There must be something wrong with our isolation of
DNA protocol. That is why we used a new protocol. With this protocol we got an
OD260/OD280 of 1.2. The ratio was low meaning that it is impure and probably does not
contain much DNA. Again, we could not properly isolate the DNA. What also is a
mystery is that our cells did not grow in the liquid LB/Kan overnight tube. I think that
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some of the solutions that we used to isolate the DNA were contaminated or at the wrong
concentration. The ethanol that was used could be at lower concentration than what is
expected. If the concentration of ethanol to wash the DNA is too low then the DNA
could dissolve in the aqueous phase of the water when the DNA is air-dried. Even
though we did get DNA most of it could have been lost somehow to the reagents that we
used. When I did a lab like this before, we got DNA and it showed up on the agarose gel
and it had a similar protocol. Next time, freshly prepared reagents should be used if this
is the case. When the cells were put in an LB/Kan media tube, it could be that the cells
could be sensitive to certain temperatures and the incubator room was not an optimal
temperature to grow at.
If we did get mutants of interest, we could isolate the transposon and clone it into
another bacteria cell, which will make more copies of it. Then we can isolate the plasmid
DNA and sequence this. When we get the results back from sequencing, we can find
where the transposon got integrated into bacteria’s DNA. Also, we can find the gene of
interest that caused its mutagenic phentype because the gene sequence surrounds the
transposon that was used. From this we can learn the nature and identity of the gene and
how it affects the cells ability to survive and grow.
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