Created: Sep 2021
The study of microbial inheritance starts with the acquisition of mutants distinguishable from other individuals. Mutations can occur naturally in the population. If these mutants can be selected successfully, there is no need to artificially induce mutations. However, in practice, it is necessary to make an appropriate selection after artificially inducing mutations to increase efficiency. We can induce mutations using physical and chemical sources. Physical mutation sources include X-ray and ultraviolet light, and chemical mutation sources include N-methyl-N-nitroso-N-guanidine (NTG) and nitrous acid. Chemical mutagens are generally carcinogenic and require careful handling.
In many cases mutated gene is repaired by the repair mechanism provided in the cell. However, if repair is not performed by expression of the SOS mechanism, the mutation stays. When a mutation occurs in a gene essential for survival, it leads to death, but when it is not a lethal mutation, new genetic traits may be acquired.
In this experiment, streptomycin (antibiotic) sensitive Escherichia coli (BL21). is irradiated with ultraviolet light for a short time to induce mutation, and E. coli that has acquired resistance to streptomycin appear. The objective of this experiment is to examine the relationship between the UV irradiation time and survival rate, and between the UV irradiation time and the appearance rate of mutant strains.
Equipment and Reagents
• E. coli preculture liquid (10 mL LB liquid medium)
• LB liquid medium (2 mL, 5 test tubes)
• LB medium plate (with streptomycin, 5 plates)
• 15 LB medium plate
• Saline solution
• Microtube 1 (for samples, 5 tubes)
• Microtube 2 (for 990 μL saline solution, 5 tubes)
• Microtube 3 (for 900 μL saline solution, 15 tubes)
UV Irradiation, Sample Collection, Dilution and Cultivation
1) Place 10 mL of culture liquid in a sterilized centrifuge tube and centrifuge for 10 min at 3000 rpm to precipitate the bacterial cell.
2) Working inside of a clean bench, remove the culture liquid from the tube and add 5 mL saline solution to the E. coli pellet. Suspend the pellet and centrifuge for 10 min at 3000 rpm again.
3) Remove the liquid from the tube, add 20 mL saline solution to the pellet and suspend it again. Pour it into a sterilized petri dish, also placing a sterilized magnetic stir bar in the dish.
4) Place a polystyrene box inside of the clean bench and put a magnetic stirrer on top of it. Put the petri dish on the stirrer. Do not cover the dish. Remove 500 μL of E. coli liquid from the dish and place it in a microtube as the 0s UV irradiation time sample. Turn off the light in the clean bench.
5) Turn on the UV light while stirring the E. coli liquid. Keep irradiating for 15s, then turn off the lamp. Collect 500 μL from E. coli liquid from the dish and place it in a microtube as the 15s UV irradiation time sample.
6) Repeat the aforementioned procedure, irradiating the bacteria with UV light for another 15s. Collect the 30s UV irradiation sample.
7) Continue irradiation and collect the 45s and 60s UV irradiation samples
8) Collect 100 μL from each Microtube 1 (0- 60s irradiation) and place it in 5 test tubes containing 2 mL of LB medium. Place it in laboratory incubator shaker at 37ºC for one night.
9) Collect 10 μL from the remaining liquids from Microtubes 1 and place it in microtubes containing 990 μL saline solution (x100 dilution). Mix well.
10) Collect 100 μL from the x100 diluted solutions and place it in microtubes containing 900 μL saline solution (x1,000 dilution). Mix well.
11) Collect 100 μL from the x1,000 diluted solutions and place it in microtubes containing 900 μL saline solution (x10,000 dilution). Mix well.
12) Collect 100 μL from the x10,000 diluted solutions and place it in microtubes containing 900 μL saline solution (x100,000 dilution). Mix well.
13) Place 100 μL of each of the 3 diluted solutions (x1,000, x10,000, x100,000 dilution) in 3 LB medium plates and apply using a spreader. There should be 15 LB plates in total, 3 per each UV irradiation time. Incubate the plates at 37ºC for one night
Mutant strain transplantation (the following day)
1) Collect 1 mL from each of the incubated cultivation test tubes (2.1.8)), place it in microtubes and centrifuge in order to precipitate bacterial cells.
2) Reduce the medium to around 150 μL, suspend it and apply to LB plate containing 100 μg/mL streptomycin with a spreader (5 LB plates in total). Incubate the plates at 37ºC for one night.
Counting the colonies (the following week)
1) Count the number of colonies on x1,000, x10,000, x100,000 dilution LB plates. For every irradiation time, choose one of the three plates that has the colonies that are easiest to count. Calculate the number of colonies for the x1 solution (e.g. number of colonies on the x1,000 dilution plate times 1,000) and present the data in a table. Find the survival rate of E. coli depending on the UV irradiation time.
2) Count the number of colonies on the LB plate containing streptomycin. Investigate the relationship between the survival rate and mutagenesis (induced mutation).
For every irradiation time, one of the three (x1,000, x10,000, x100,000 dilution) LB plates was chosen and the number of colonies was counted. To make it easier to compare the data, the (theoretical) number of colonies for undiluted (x1 dilution) solution was calculated.
The survival rate of colonies for 0s irradiation was assumed to be 100%. Using the value of the number of colonies at 0s, survival rate was calculated. The results are displayed in Table 1. In addition, the irradiated E. coli was grown on LB plates containing streptomycin to see if UV light induced a mutation that would make the bacteria immune to this antibiotic. The results are displayed in Table 2.
Moreover, using the Colony number for x1 solution data from Table 1 and Colony number from Table 2, mutation rate for the highest survival irradiation time (15s) has been found to be 4.07 × 10-4 %. In case of 30s irradiation, the mutation rate is 4.69 × 10-4 %.
As can be seen from Table 1, the survival rate decreases sharply between 0s and 30s irradiation. This suggests that even due to a short exposure to UV light, most of the E. coli develop a mutation in a gene that is crucial for their survival, which results in death of those cells. However, there is not much decrease in survival rate between 45s and 60s irradiation. Although by that point only a fraction of the original E. coli population remains, that fraction consists of the strongest individuals. Perhaps their genes differ slightly from that of the most BL21 E. coli cells, making them less susceptible to damage from UV light. For example, they could have a particularly effective SOS mechanism that keeps repairing the mutations caused by irradiation.
The number of colonies formed by E. coli grown on LB medium containing streptomycin (as opposed to LB medium only) was even lower. This is because, in this case, E. coli would have to both withstand the UV irradiation and develop a mutation that makes it immune to streptomycin in order to survive. Based on the experimental results, E. coli are most likely to fulfil those 2 conditions if they are subjected to UV light for 15s. Nevertheless, even in this case the mutation rate (gaining streptomycin immunity) is as low as 4.07 × 10-4 %.
While the overall number of colonies for 30s irradiation time was only 1, the mutation rate turns out to be higher than for 15s irradiation and is equal to 4.69 × 10-4 %. This is because between 15s and 30s irradiation the LB only Colony number for x1 solution (Table 1) decreased 8 times, while the colony number (Table 2) decreased 7 times.
While the optimum irradiation time for streptomycin immunity induction has been established using the experimental results, this value is only an approximation. It is possible that there would be more colonies developing on streptomycin LB plates for the irradiation time close to, but not equal to 15s. To obtain a more accurate value of optimum irradiation time, the samples for cultivation could be collected every 5 seconds within the 0-30s range.
As the result of irradiating the BL21 strain of E. coli with UV light and cultivating the resultant samples on LB only medium plates, there was considerable decrease in survival rate of the bacteria, which demonstrates the disinfecting properties of UV light. When the irradiated bacteria were grown on LB plate containing streptomycin, the colony number diminished further, the highest number of colonies appearing for the sample irradiated for 15 seconds. By investigating both the relationship between the UV irradiation time and survival rate, and between the UV irradiation time and the appearance rate of mutant strains (mutation rate), the objective of this experiment has been fulfilled.