Genetics And Cell Biology Laboratory Biology Essay

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In this experiment, mutation in E. coli bacterial cells is learned. Mutations occur when DNA sequence are altered: either deletions or insertions of a single or multiple base pair that can lead to frameshift mutations. Mutation frequency measures how frequently the cells are mutated in a given population. Spontaneously occurring mutation is not frequent (10-5 to 10-10 mutation frequency).2) Therefore, to induce the mutation, it would be necessary to use chemical mutagens. It incorporates into DNA as adenine or guanine leading to mispairing of guanine; AT to GC transition and GC to AT can occur as a consequence of 2AP induced mutation. It is relatively weak than other chemical mutagen and the procedure is simple to mutagenize E. coli with 2AP. Therefore, it is relevant to use 2AP. E. coli was exposed to 2-AP and the effect on E. coli by 2-AP was observed by comparing with E. coli grown without 2-AP. In the second part of the experiment, insertional mutagenesis of Tn10 are used. (1)

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E. coli strain used was CC102 (ara ∆(gpt- lac)5 thi rpsL/F'lacZ- Y+ A+ proA+B+).(refer) This strain is auxotrophic: it requires thiamine, is streptomycine resistant and it is missing Lac Z gene. CC102 is missing LacZ, therefore, when the mutation occurs by 2AP, it could gain the LacZ function by mutation. The mutation is done in random locations. The mutation can be tested by selection, the growth condition in which only a specific type of strains can grow. Afterwards, it can be tested by screening with IPTG and X-gal containing plate where it would show blue when Lac Z is active. However, this screening method is not used in this experiment. Instead, replica assays are made to identify auxotrophic mutants. Since auxotrophic mutants will not grow on minimum glucose, the number of auxotrophic mutants can be identified by comparing the colonies in "LB tet" medium containing plate to the colonies in minimum glucose. In order to determine mutation frequency, viable count from the LB plate is used as number of colony formed in a plate and number of colony grown in different media are used to determine the mutation frequency. (1)(2)

Insertional mutagenesis is done by Tn10. Tn10 is a transposon that carries tetracycline resistance gene. For this to be inserted to the cell, lambda phage was used. To prevent the lambda phage from lysogenizing, its attachment site was eliminated and was replaced with Tn10. Then, site specific recombination would not occur. Tn10 would be successfully inserted by lambda phage and create random mutations. Also, the mutated cell will now be resistant to tetracycline. (1)

Material and Methods:

Please Refer to Biol 368 lab manual*

All procedures are performed according to the BIOL 368 lab manual (Concordia Biology Department 2010) except the followings: in part A, step 6, we added 1.0 ml of Tryptone broth containing sodium citrate to each tube instead of 1.8 ml. Also, for the section data of part B, given data is used instead of our actual section data.

Results:

Part B:

Table 1: Viable count and Mutation Frequency for CC102 (Group 4) (with and without 2-AP treatment)

Medium

Culture Dilution

2-AP Treatment

# of Colonies

Viable count (cfu/ml)

Frequency (cfu)

Tet

Undiluted

+

0

N/A

<4.55x10-8

-

9

N/A

<1.23x10-8

Nal

Undiluted

+

38

N/A

1.73x10-6

-

2

N/A

2.47x10-8

Lac

Undiluted

+

0

N/A

<4.55x10-8

-

0

N/A

<1.23x10-8

LB

10-5

+

259

2.59x108

N/A

10-6

22

2.20x108

N/A

10-5

-

550

5.50x108

N/A

10-6

81

8.10x108

N/A

Table 2: Viable Count and mutation frequency for the Thursday Section (NO 2-AP Treatment)

Viable Count (cfu/mL) in following condition

Mutation Frequency (cfu) in following medium

Group Number

LB (10-5)

LB (10-6)

LB Tet

LB Nal

Lac

1

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N/A

1.69E+09

6.39E-07

8.28E-08

4.14E-08

2

4.28E+08

4.80E+08

2.20E-08

4.41E-08

1.18E-08

3

N/A

1.68E+09

2.14E-07

1.19E-08

2.20E-08

4

5.50E+08

8.10E+08

1.47E-08

2.94E-08

1.19E-08

5

2.26E+09

1.20E+10

3.37E-08

1.55E-05

6

N/A

4.90E+09

2.20E-07

8.16E-09

9.81E-09

7

3.39E+08

2.80E+08

3.23E-08

3.23E-08

4.08E-09

8

5.60E+08

1.70E+09

8.85E-09

1.77E-08

3.23E-08

9

3.36E+08

6.30E+08

2.07E-08

2.07E-08

8.85E-09

10

N/A

4.80E+08

4.17E-08

4.17E-08

2.07E-08

Average

7.46E+08

2.47E+09

1.35E-07

3.22E-08

1.57E-06

Standard Deviation

6.85E+08

3.43E+09

1.95E-07

2.034E-08

4.64E-06

*Added for project4 2nd half: group 5 on Lac plate gives an outlier:

Grubbs test: Z= = 3.00 -> not acceptable

Without group 5 data:

Average mutation on Lac plate: 1.81E-08

Table 3: Viable Count and mutation frequency for the Thursday Section (2-AP Treatment)

Viable Count (cfu/mL) in following condition

Mutation Frequency in following medium

Group Number

LB (10-5)

LB (10-6)

LB Tet

LB Nal

Lac

1

7.00E+06

2.00E+07

N/A

1.48E-06

3.70E-06

2

3.05E+08

3.10E+08

3.25E-08

1.53E-06

4.88E-07

3

2.83E+08

N/A

1.10E-05

3.53E-07

3.53E-07

4

2.59E+08

2.20E+08

4.18E-08

1.59E-06

4.18E-08

5

2.20E+07

N/A

2.73E-06

1.09E-05

N/A

6

5.00E+06

2.00E+07

N/A

8.00E-07

1.60E-06

7

4.02E+08

4.30E+08

2.40E-08

1.20E-06

1.44E-07

8

2.24E+08

1.94E+09

9.24E-09

5.18E-07

3.70E-08

9

2.75E+08

3.60E+08

3.15E-08

6.30E-07

3.15E-07

10

4.91E+08

4.30E+08

2.17E-08

3.04E-07

1.74E-07

Average

2.27E+08

4.66E+08

1.73E-06

1.93E-06

7.62E-07

Standard Deviation

1.59E+08

5.78E+08

3.60E-06

3.03E-06

1.13E-06

Sample Calculation

Viable count with 2AP treatment of group 4 in LB (10-5 diluted)

Mutation frequency in tet medium:

*when the mutant colonies were 0, we considered it as 1 (will be discussed in discussion)

Identifying Lac+:

= viable count x volume x dilution factor = 2.40 x 107

Probably of finding mutant:

10-5 plate:

Probability of finding mutant:

Part A:

Table 4: Raw data of my group (group 4)

Tn10 Insertion to D10

Undiluted

10-1

10-2

10-3

#colonies

12

49

35

14

Mutation frequency

8.00E-8

3.27E-7

2.33E-7

9.33E-8

Table 5: Section data for mutation frequencies (section 3)

Group Number

Mutation Frequency (Undiluted)

Mutation Frequency

(10-1)

Mutation Frequency

(10-2)

Mutation Frequency

(10-3)

1

7.33E-08

2.67E-07

7.33E-08

6.67E-09

2

1.13E-07

3.13E-07

1.27E-07

2.00E-08

3

1.13E-07

4.13E-07

1.33E-07

6.67E-09

4

8.00E-08

3.27E-07

2.33E-07

9.33E-08

5

1.20E-07

4.13E-07

1.73E-07

1.33E-08

6

6.67E-08

3.53E-07

1.07E-07

6.67E-09

7

1.20E-07

3.07E-07

5.33E-08

6.67E-09

8

1.20E-07

3.73E-07

1.40E-07

6.67E-09

9

7.33E-08

2.87E-07

1.13E-07

1.33E-08

10

1.07E-07

1.15E-06

6.00E-08

6.67E-09

Average mutation frequency of the section

9.87E-08

4.20E-07

1.21E-7

1.80E-8

Standard Deviation of the section

9.87E-08 ±

2.24E-8

4.20E-07 ±

2.6E-7

1.21E-7 ±

5.45E-8

1.80E-8 ±

2.69E-8

Table 6: Raw data of Replica Plates

Colonies count on LB Tet Plate

Auxotrophic colonies count

40

0

Auxotrophic Mutation Frequency Calculation:

0/40 = 0

Table 7. Replica plate results and the mutation frequency in section

Group

Colonies on LB Tet Plate

Auxotrophic Mutant colonies

Auxotrophic Mutation Frequency

1

9

1

1.11E-01

2

29

56

1.93E+00

3

56

29

5.18E-01

4

40

0

0

5

62

2

3.23E-02

6

48

40

8.33E-01

7

52

0

0

8

27

34

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1.26E+00

9

43

43

1.00E+00

10

35

0

0

Section Average

40.1

14.375

5.21E-01

Standard Deviation

40.1±

14.94289

14.375±

20.9392

5.21E-01±

0.637093

*Since group 2, 8 data contains of contamination, it is excluded to calculate average and S.D.

(group 2 contains a lot of contamination)

Discussion:

In part B, 2-AP, a chemical mutagen, was added to induce the frequency of the mutants. As the table 4 shows 2-AP did induce the mutation frequency. The Tet plate and Nal plates contain tetracycline and nalidixic acid respectively. In Nal plate, there were 38 colonies found with 2-AP and 2 colonies without 2-AP treatment. The mutation frequency was found to be 1.73x10-6 and 2.47 x10-8 respectively. With 2-AP treatment, we have almost 100 times higher mutation frequency. Tet plate had 9 colonies of CC102 cell without 2-AP and 0 colonies with 2-AP treatment. For this, there are two possibilities. Some of the C102 cells might have had some tetracycline resistance element initially, but reverse mutation occurred, and the cells lost the tetracycline resistance element. Otherwise, the 9 colonies could have been just contamination. Or it could be possibly because the 2-AP did not affect positively as it did with the Nal plates. Either way, in Nal plate, there is a significant change in mutation frequency whereas in Tet plate, it does not show much of difference in mutation frequency. Looking at the Lac plate, with and without 2-AP treatment, both did not have any colonies. CC102 initially lacks lac Z gene. Thus, reverse mutation is necessary to have lac Z+ and be able to grow on the lac plate. However, the mutation occurs randomly, and it happens that the reverse mutation did not occur. This random mutation occurs due to the absence of the attachment site. Now looking at the section data, without 2-AP treatment, we have mutation frequency of 1.35*10-7, 3.22*10-8 and 1.57*10-6 for Tet, Nal, and Lac plate respectively. With 2-AP treatment, we have 1.73*10-6, 1.93*10-6 and 7.62*10-7 for Tet, Nal, and Lac respectively. So, the mutation frequency increased by around 10 for Tet, 100 and for Nal, and decreased by 10 for Lac plate. As our group data represents, Nal plate have a biggest difference in colonies with and without 2-AP. This means that 2-AP induces better in Nal medium rather than in Tet medium. For Lac plate, there is an outlier on group 5. Excluding that value gives an average of 1.81*10-8. This means that on the Lac plate, the mutation frequency increased by about 100. From this, we can deduct that our group data for Lac may be wrong, or happened by possibility. Also, 2-AP induces well in Lac medium as much as in Nal medium.

Cupples and Miller did a similar experiment using 2-AP on 6 E. coli. They measured the frequency of the reversion mutation in lacZ gene. Eventually, the frequencies of revertant increased as more and more of 2-AP are introduced to CC102. Their result says that 2-AP induced G or C transformed into A or T. This resulted in transforming of the codon at 461 that makes the cell lac Z+ (more specifically GGG to GAG). The maximized mutation was found when 700 μg/ml was used. There were 457 colonies found with 700 μg/ml whereas, when 10 μg/ml of 2-AP was used, only 31 colonies were found. Therefore, 2-AP does induce the mutation.

In part A, Tn10 was inserted by using lambda phage to the bacterial chromosome. D10 was used as a model. D10 cell becomes tetracycline resistant when Tn10 is properly inserted to the chromosome because Tn10 contains tetracycline resistance element. Screening was done to observe mutant phenotypes. To do this, one of the plate where Tn10 was inserted was used as a master plate to replicate on LB Tet plate and minimum glucose plate. Since auxotrophic mutant would not grow on minimal medium, we can simply see where the colonies did not grow comparing with LB Tet plate. As a result, we had 40 colonies on the LB Tet plate and no auxotrophic mutation was found, or 40 colonies were found on minimal glucose plate. This result is as expected. It eventually grew on LB Tet plate since Tn10 was inserted, meaning that the insertion was done successfully. Then, the same number of colonies were found in the minimal plate. This is due to the fact that the mutation did not interrupt any of the gene that is necessary to metabolize glucose. Thus, auxotrophic mutant was not found in our plate. This result is normal because only 1% of the colonies are expected to be auxotrophs (2). Our probability of finding mutant is calculated to be 1.09 * 10-5, so it is less than 1%. As a result we earned 0 auxotrophic colonies, and therefore, the prediction is correct.