Download Experiment 3

Bryan Fong
MIC 155L
Experiment 3: Mutagenesis, Selection, and Screens
Abstract
In this lab we tried to induce mutations in E. coli cells using specific mutagens.
The mutagens used were EMS, UV, NG, and EtBr. In our group, we used EMS. We
took different concentration of EMS and exposed it to E. coli cells and then plated this
for a viable count. The resulting viable count that was determined was used to make a
kill curve, which we used as a standard. This standard was used to determine a range of
concentration where would get 90-90% killing of the bacteria. After finding out the
concentration range we exposed E. coli to the EMS at this new range and plated for
viable cell count. We also screened the bacteria for Lac- and StrpR mutations by growing
them on tetrazolium plates and LB strep plates. For our results, we got Lac- mutants but
no StrpR mutants.
Introduction
According to the literature, a mutation that results in a lac- phenotype will
naturally occur in one of 107 cells. For a strepR mutation to occur, a mutation will occur
naturally in one of 109 cells. To get any one of these mutations one must need to start out
with a lot of cells to have chance isolating these mutants. To increase the frequency of
mutants, we introduce the cells to mutagens. Mutagens cause mutations by either by
chemical or irradiation that damage DNA. Depending on what type of mutagen there can
variety of effects that it could do on the cells DNA; base substitutions, deletions,
frameshifts, transitions, transversions, etc.
To get a general idea of what range of mutagens are effective in producing the
mutants that we want, a “kill curve” is made up. A “kill curve” is either the number of
cell survivors vs. time of mutagen exposure or number of cell survivors vs. concentration
of mutagen exposed to the cells. After the cells are exposed to the mutagen, the cells are
plated for a viable cell count to see the number of survivors. Ideally 90-99% killing is the
range of mutagen that we want to find the mutants. The survivors of the 90-99% percents
will have the highest probability of having the mutant of interest.
Finding out that we have 90-99% killing, we want to plate the survivors for a
viable cells count. The cells are plated on selective media that we allow the phenotype of
the mutation of interest. After that we can screen the possible mutant to see if we truly
got the mutant that we are interested in.
In our experiment, we were assigned the mutagen EMS (ethyl methane sulfonate).
EMS along with other similar mutagens are alkylating agents which directly modify the
bases of the DNA by adding alkyl groups to the nitrogen ring. This could lead to
mismatching of base pairs during DNA replication or errors during DNA repair. It is
found that EMS can cause transitions and transversions. Knowing that we can get these
type mutations, I believe EMS can induce a fair amount of mutations that we are
interesting because EMS will either alter or inactivate the genes of the wild phenotype.
Methods
Part I: Kill curves
Ten milliliters of a fresh culture of Escherichia coli MG1655 in M9 was spun
down for 10-20 minutes in the centrifuge. After the spin, the pellet was re-suspended in
1ml of 1x M9 medium. Then, 100ul of the re-suspended culture was added to 6 different
cultures containing 900ul of M9 medium. .001ml, .005ml, .01ml, .02ml, .04ml, and
.08ml of EMS were added to the test tubes. The test tubes were aerated for 2 hours in the
37 C bath. After the incubation period, the samples were washed twice by spinning them
in the microfuge for 30 seconds and re-suspending the pellets in 1x M9. After the second
wash, the pellet was re-suspended in 100ul of 1x M9 medium. The 100ul was used to set
up a 8-fold serial dilution. The dilutions were plated on LB plate for viable count. The
plates were incubated in the 37 C for 24 hours.
Part II: Mutagenesis and identification of Lac- and StrpR cells
A protocol was designed to achieve 90% killing based on the results from the
generic kill curve that was conducted from part I.
Thirty milliliters of a fresh culture of E. coli MG1655 in M9 was spun down for
10-20 minutes in the centrifuge. After the spin, the pellet was re-suspended in 600ul of
1x M9. Then 100ul of the re-suspended sample was added to 6 test tubes containing
900ul of M9 medium. 10ul, 15ul, 20ul, 25ul, 30ul of EMS was added to each test tube.
The test tubes were aerated for 2 hours in the 37 C bath. After the incubation period, the
samples were washed twice using by spinning them in the microfuge for 30 sec and resuspending the pellets in 1x M9 medium. After the second spin, the pellet was resuspended in 100ul of 1X M9 medium. The 100ul was used to set up a 8-fold dilution.
The dilutions were plated on LB plates for viable count, on LB with Tetrazolium plates to
screen for Lac- mutants, and also on LB strp plates to select for StrpR mutants. The
plates were incubated in the 37 C room for 24 hours.
After the incubation period, Lac- and StrpR mutant colonies were isolated and
purified by streaking them on LB plates and incubating them in the 37 C for 24 hours.
The next day, the colonies from the Lac- and StrpR mutants on the LB plates were
streaked onto LB with Tetrazolium plates and LB strp plates to confirm that they are Lacand Strp R mutants. The plates were incubated in the 37 C and incubated for 24 hours.
Then the next day overnight liquid cultures of the Lac- and StrpR mutants were made and
incubated in the 37 C. Also the mutant colonies were streaked on MacLac , MacMal, and
MacAra plates and incubated for 24 hours in the 37 C room. The following day, freezer
stocks of the liquid cultures were made.
Results
Mutagen
1 EMS
Dosage/Time
15ul
20ul
30ul
40ul
2 EMS
10ul
15ul
20ul
25ul
30ul
3 25ug/ml NG 15 min.
4 25ug/ml NG 10 min.
15 min.
20 min.
25 min.
5 25ug/ml NG 2.5 min.
5 min.
10 min.
20 min.
30 min.
6 EtBr
Lac1
2
2
2
0
0
0
1
2
2.00E+04
1.00E+03
1.10E+03
6.00E+02
1.00E+02
200
2800
220
24
5
0
0
StrpR
Total # Plated Total #
Survivors
0
3.26E+09
1.31E+09
0
3.26E+09
5.50E+08
0
3.26E+09 >5E+08
0
3.26E+09
1.24E+08
0
1.92E+09
1.19E+09
0
1.92E+09
7.50E+08
0
1.92E+09
8.50E+08
0
1.92E+09
4.90E+09
0
1.92E+09
4.90E+07
0
2.17E+07
3.00E+06
0
6.00E+06
3.00E+06
0
6.00E+06
2.10E+06
0
6.00E+06
1.40E+06
0
6.00E+06
1.20E+06
0
1.10E+09
9.25E+08
0
1.10E+09
3.00E+06
0
1.10E+09
3.40E+05
0
1.10E+09
1.00E+05
0
1.10E+09
4.00E+03
201
8.00E+08
8.00E+08
25ug/ml
50ug/ml
75ug/ml
100ug/ml
7 EtBr
0
25ug/ml
50ug/ml
75ug/ml
100ug/ml
8 EtBr
9 UV
10 UV
0
25ug/ml
50ug/ml
75ug/ml
100ug/ml
150ug/ml
20 J/m2
40 J/m2
60 J/m2
80 J/m2
100 J/m2
20 J/m2
40 J/m2
60 J/m2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
165
235
190
7
230
277
85
118
35
58
35
12
7
16
0
1
0
0
0
0
0
0
8.00E+08
8.00E+08
8.00E+08
8.00E+08
3.46E+08
3.46E+08
3.46E+08
3.46E+08
3.46E+08
6.30E+08
6.30E+08
6.30E+08
6.30E+08
6.30E+08
6.30E+08
1.73E+09
1.73E+09
1.73E+09
1.73E+09
1.73E+09
8.00E+07
8.00E+07
8.00E+07
5.10E+08
5.90E+08
3.60E+08
8.30E+07
2.65E+09
3.00E+08
3.00E+07
2.50E+07
2.00E+07
1.16E+09
7.95E+08
4.25E+08
6.10E+08
5.50E+08
2.99E+09
3.94E+07
4.31E+06
2.87E+06
3.32E+06
3.18E+07
7.10E+06
1.60E+06
Discussion
From our data and our kill curve we got 90-99% killing of bacteria between 20-30
μl of EMS. The mutations of interests should be found in this range. As we suspected,
we found possible Lac- mutants in the form of pink colonies on the tetrazolium plates,
but there were no strpR that grew on the LB strp plates. The pink, possibly Lac- mutants
were taken and purified on LB agar plates. The purified colonies were then plated on
tetrazolium, MacLac, and MacAra agar plates. Resulting from the tetrazolium plate the
colonies came out top be red indicating that we have Lac- mutants. On the Macconkey
agar plates, there were white colonies on the MacLac meaning that we have Lac-mutants
because they could not utilize the sugar lactose. On the MacAra agar plates, the colonies
were red meaning that we did not get an Ara- mutation from our mutagenesis.
We did not get as many mutants as we hoped from out mutagen. Perhaps we
should have extended the increments of EMS dosage from 20, 25, and 30 μl to 20, 22.5,
25, 27.5, and 30 μl or used a higher amount of bacteria cells from the start of the
experiment. Most of the mutations that occurred may have only been silent mutations so
nothing has changed and got few Lac- bacteria out of the total population.
From the data it seems as though UV is a bad mutagen because there were one or
two mutants. UV causes a number of different mutations that can easily produce
mutants, so I think that the concentration of UV light was not high enough to induce
mutations. The EtBr got no Lac- mutants, but they did get quite a few strepR mutants.
Known mainly to cause frameshifts, it would seem more likely to get strepR depending
where EtBr affects the DNA. I think that the EtBr affects the regions of DNA where
there the strepS gene is and with the deletions and insertion it could cause the DNA to
allow resistance to streptomycin. Therefore EtBr is possibly a good mutagen to get
strepR mutants. In contrast, Nitrosoguanidine (NG) causes base substitutions usually
around the Lac operon. Since there were many Lac- mutants, then that justifies that NG
is a powerful mutagen to get Lac- mutants.
Besides EMS and UV, the mutagenesis of the E. coli turned out good. We now
know what mutagens to use if we want Lac- or strepR bacteria. For EMS, next time the
dosage range should be extended in smaller increments between 20-30 μl and/or use a
higher cell concentration initially. Also we can start predicting where the mutations have
occurred in the Lac operon like in the operator, promoter, CAP-Camp regions of the
DNA. We can use sequencing to figure this out.