MOLECULAR GENETICS MUTATION LABORATORY PRACTICALS REPORT

Introduction

Molecular genetics is concerned with genes function and structure at molecular level. Genetics are the determinants of heredity, but they are subject to variations as a result of mutations. Therefore, molecular genetics mutations are the alterations of genes occurring at molecular level. Therefore, the bacteria reversed mutation assay is utilised in the evaluation of the mutagenic properties of various materials. This test uses strains of Salmonella typhimurium that are amino acid-dependent. In the absence of histidine which is externally provided, the cells can’t grow continuously leading to formation of colonies. However, colony growth resumes if a reversion mutation reversion occurs, allowing a resumption of histidine production. This implies that different concentrations of histidine and biotin can be used for selective growth of these bacterial strains. The occurrence of spontaneous reversions is usually with of the strains; this is due to the fact that mutagenic compounds result to increased number of reverting colonies. Thus, the reversion rate for any mutant bacterial strain can be determined.

A mutation in the DNA repair (uvrA/B) leads to elimination of the excision repair, a DNA repair pathway for UV light induced damages/mutations as well as certain mutagens. When the uvrA/B mutation is present in a bacterial strain it makes it more sensitive to mutagens as well as UV light. Moreover, the uvrA/B mutation involves a section of deletion mutation that extends into the biotin synthesis gene; therefore, the requirement for biotin is caused by the deletion of the genetic region concerned with biotin synthesis. Therefore, an indication of uvrA/B mutation is increased sensitivity to UV light. Furthermore, the rfa mutation results to a change in the bacterial cell wall properties resulting to partial loss of the barrier of lipopolysaccharide (LPS) thereby leading to increased cells’ permeability to certain types of chemicals including dyes such as crystal violet and sodium deoxycholate. Thus, an indication of rfa mutation is the increased sensitivity these dyes. These laboratory experiments have sought to investigate these biological concepts in a series of four experiments where the first three are grouped into practical 1 while the remaining experiment 4 is grouped into practical 2. The findings of these experiments are presented in this laboratory report write up.

Objective

The main objective of these laboratory experiments was to undertake the testing of Salmonella typhimurium strains for sensitivity to mutation as well as investigating of the effect of known mutagens on the reversion rate of three auxotrophic strains of Salmonella typhimurium such as TA1535, TA1537 and TA1538.

Methods

Materials and apparatus

Practical 1(Experiment 1, 2 and 3)

1. Molten      agar
2. Waterbath
3. Bacterial      culture
4. Gilson      pipette with sterile tip
5. E      medium plate
6. 0.5      mM Histidine solution
7. 0.1      M Histidine solution
8. 0.5      mM Biotin solution
9. ¼      strength Ringer’s solution
10. Eppendorf      tube
11. Marker      pen
12. Glass      spreader
13. Nutrient      agar plate
14. Crystal      violet solution
15. Sodium      deoxycholate solution
16. Sterile      inoculation loop
17. Filter      paper
18. Sterile      forceps
19. Paper      discs

Practical 2 (Experiment 4)

1. Petri      dishes
2. Marker      pen
3. Sterile      centrifuge tubes
4. Waterbath
5. Molten      agar
6. 0.5      mM Histidine solution/0.5 mM Biotin solution
7. Mutant      bacterial cultures i.e. TA 1535, TA 1537 and TA 1538.
8. EMS
9. DAA

Procedure

Practical 1(Experiment 1, 2 and 3)

Experiment 1: Effect of Histidine on Growth

 

This experiment tested the requirements of histidine by the auxotrophic mutant strains as well as their spontaneous reversion rate and our Group carried out the experiments using the mutant strain TA 1537.  The procedure was as follows:

 

1. Growth on medium E

 

(i)              2mL of molten overlay agar (kept in a 450C waterbath) were added in to a sterile tube. This was followed by a waiting period until the molten agar cooled slightly (to about body temperature (370C), in order to ensure the bacterial strains were not killed).

(ii)            0.1 mL of the bacterial culture were added using a Gilson pipette fitted with a sterile tip.

(iii)          The molten agar was immediately poured along with the bacterial strain onto an E medium plate making sure that the overlay agar was evenly distributed on the plate surface.

(iv)          The plate was allowed to cool and later incubated at 370C overnight.

 

 

2. Growth on medium E supplemented with low concentration of histidine      and biotin

 

(i)              2mL of molten overlay agar were added in to a sterile tube followed by a period of waiting until it slightly cooled, 0.2mL 0.5mM histidine + 0.5mM biotin solution were then added using sterile tips.

(ii)            0.1 mL of the bacterial culture were added using a Gilson pipette fitted with a sterile tip.

(iii)          The molten agar was immediately poured along with the bacterial strain onto a E medium plate taking in order to ensure that the overlay agar was evenly distributed on the plate surface

(iv)          The plate was then allowed to cool and later incubated at 370C overnight.

 

3. Growth on medium E supplemented with high concentration of histidine      and biotin

 

(i)              2mL of molten overlay agar were added in to a sterile tube followed by a period of waiting until it cooled slightly; in addition, 0.2mL 0.5mM histidine + 0.5mM biotin solution and 0.1mL of the 0.1M histidine solution were added using sterile tips.

(ii)            The bacterial culture was not added to this tube.

(iii)          The molten agar was immediately poured onto an E medium plate carefully in order to make sure evenly distributed overlay agar on the plate surface.

(iv)          The plate was then allowed to cool.

(v)            Six serial dilutions of the bacterial culture in ¼ strength Ringer’s solution (10-1, 10-2, 10-3, 10-4, 10-5 and 10-6) were then prepared; this was done by adding 900ml of  ¼ strength Ringer’s solution in a sterile eppendorf tube and adding 100ml of the bacterial culture in order to make 10-1 dilution. This mixture was then followed by vortex to ensure thorough mixing. The diluted culture was used to carry out the next dilution and this procedure was repeated six times in order to make sure a 10-6 dilution was obtained). The plates were waited to set and dry (for step number VI).

(vi)          A marker pen was used to divide the plate into two halves and the plate’s bottom was marked (as shown in Figure 1 below). One side was marked 10-5 while the other side was marked 10-6. 10ml of the 10-5 dilution were dropped onto the plate using a Gilson pipette and a glass spreader was used to spread it on one side of the plate while 10ml of the 10-6 dilution were dropped and spread with a glass spreader on the remaining side of the plate.

(vii)        The plates were then overturned and sealed with parafilm followed by the labelling of the plates with the group members’ names and the bacterial strain used. The plate was then incubated at 37oC for 24hrs.

 

Figure 1: 10-5 and 10-6 dilution marked plate

10-6

10-5

 

 

 

 

 

 

 

 

 

 

 

Experiment 2: Sensitivity of Strains to Crystal Violet and Sodium Deoxycholate

This experiment aimed at testing the lack of the lipopolysaccharide layer in the mutant strain TA1537 because the lipopolysaccharide layer usually acts as a barrier to sodium deoxycholate and crystal violet. Hence, the mutant strains lacking this lipopolysaccharide layer tend to be sensitive to sodium deoxycholate and crystal violet. Our group carried out the experiment using the bacterial mutant strain TA 1537 and the produce was as follows:

(i)              One nutrient agar plate was collected.

(ii)            2mL of molten overlay agar were added in to a sterile tube followed by a period of waiting until it slightly cooled (to about 370C (body temperature), in order to ensure bacterial strains were not killed).

(iii)          0.1 mL of the bacterial culture was added into the nutrient agar plate using a Gilson pipette fitted with a sterile tip.

(iv)          The molten agar was immediately poured along with the bacterial strain onto the nutrient agar plate carefully so that the overlay agar was evenly distributed on the plate surface.

(v)            The molten agar was then allowed to set.

(vi)          Sterile forceps were used to place two paper discs onto the agar plates surface (as shown in Figure 2 below).

(vii)        The Gilson pipette was also used to add 10ml of crystal violet solution onto one paper disc and 10ml of the sodium deoxycholate solution on the other paper disc.

(viii)      The added solutions were allowed to dry. The nutrient agar plate was then overturned and sealed with parafilm followed by labelling with group members’ names and bacterial strain used. It was then incubated at 37oC for 24 hours.

 

Figure 2: Nutrient agar plate with paper discs placed onto it

Deoxycholate

Crystal violet

 

 

Experiment 3: Sensitivity of the Strains to Ultraviolet Light

 

The deletion of uvrB prevents the functioning of the DNA repair system, hence the bacterial mutant strains becomes sensitive to UV light. This was tested in this experiment using the procedure below:

 

(i)              A line was drawn down at the bottom centre of the nutrient agar plate in order to ensure that, the agar plate is divided into two equal halves (as shown in Figure 3 below). One side of the nutrient agar plate was labelled +UV while the other side was labelled –UV.

(ii)            Another line that is perpendicular to the first line was also drawn (as shown in Figure 3 below).

(iii)          A sterile inoculation loop was used to streak the bacterial culture across the plate along the perpendicular line, and the streak was allowed to dry.

(iv)          Half of the plate was exposed to the UV light for 1 minute. This was done by covering the other half of the nutrient agar plate with cut filter paper.

(v)            The nutrient agar plate was then overturned and sealed with parafilm followed by labelling with group members’ names and bacterial strain used. It was then incubated at 37oC for 24 hours.

 

Figure 3: Bacterial culture streak on nutrient agar plate exposed to UV light

+UV

-UV

Bacterial strain

 

 

 

 

 

 

 

 

 

 

 

 

Practical 2 (Experiment 4): The effect of three known mutagens on S. typhimurium strains

The aim of this experiment was to investigate the effect of known mutagens on the reversion rate of three auxotrophic strains of Salmonella typhimurium such as TA 1535, TA 1537 and TA 1538. The procedure followed the steps below:

1. 9      petri dishes were labelled on the base A-I. The group name was also written      on the base of each plate.
2. 9      sterile centrifuge tubes were labelled A-I, and then placed into a water bath.
3. 2mL      of molten agar were added into each of the labelled tube while in the 45oC      water bath.
4. The      tube labelled ‘A’ was removed from the water bath and 200 mL      of 0.5mM histidine/0.5mM biotin solution were rapidly added, 100 mL      of the first mutant bacterial culture TA1535 were added followed by      addition of100 ml      of EMS into the tube. However, addition of EMS was done with a lot of care      because it is a potent mutagen. Molten agar was quickly swirled and poured      onto the Petri dish labelled ‘A’ which was then left to set.
5. The      tube labelled ‘B’ was removed from the water bath and 200 mL      of 0.5mM histidine/0.5mM biotin solution were rapidly added, 100 mL      of the first mutant bacterial culture TA1537 were added followed by      addition of100 ml      of EMS into the tube. However, addition of EMS was done with a lot of care      because it is a potent mutagen. Molten agar was quickly swirled and poured      onto the Petri dish labelled ‘A’ which was then left to set.
6. The      tube labelled ‘C’ was removed from the water bath and 200 mL      of 0.5mM histidine/0.5mM biotin solution were rapidly added, 100 mL      of the first mutant bacterial culture TA1538 were added followed by      addition of100 ml      of EMS into the tube. However, addition of EMS was done with a lot of care      because it is a potent mutagen. Molten agar was quickly swirled and poured      onto the Petri dish labelled ‘A’ which was then left to set.
7. The      same procedure was repeated for tubes labelled ‘D’, ‘E’ and ‘F’ for each      bacterial mutant strain such as TA 1535, TA 1537 and TA 1538, but in this      case 2-AminoAcridine, 2AA, a potent mutagen was used for each of the three      steps. Hence, this was a repetition of steps 4 to 6, where the only      difference is the mutagen used which is 2-AminoAcridine, 2AA, a potent      mutagen.
8. Furthermore,      the same procedure above was repeated for tubes labelled ‘G’, ‘H’ and ‘I’      for each bacterial mutant strain such as TA 1535, TA 1537 and TA 1538, but      in this case no mutagen was added for each of the three steps. Thus, this      was a repetition of steps 4 to 6, where the only difference is that no      mutagen was used.
9. All      the plates were then set aside in order for them to set for 20minutes
10. The      plates were then incubated at 37oC for 48 hours.
11. The      number of colonies on the plates was counted on the 13th of      November 2012.