Serial Dilutions Lab Report
1. Introduction: The goal of this lab was to demonstrate the microbiology technique of serial dilutions and how they can effectively be used in experiments. E. coli cells were exposed to UV light for various amounts of time in order to asses the effect on growth and thereby mutations since this cell was modified to not have photolyse no uvr genes for DNA repair and thus can only use the SOS response. Each group then diluted the cells accordingly based on their UV exposure time and counted the cells the next day. The counted colonies then allowed for back calculations to find the initial number of cells which then allowed for plotting of survival curves based on class data. E. coli cells were also investigated for the effect of cell number or concentration and beta-galactosidase induction. A culture of cells was grown over time
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and the lac operon was induced, the absorbance values were then used to plot growth vs. time. The amount of beta-galactosidase production of the potential mutant was then evaluated by calculating miller units which corresponds to b galactosidase production. The clas data miller units were then aggregated and evaluated to discover the identity of the mutant.
2.
UV Exposure and Colonies Observed
Fraction of Viable Cells and Time
E. coli W3110 was used as the bacteria for the UV exposure portion of the experiment as it has both mutations in the uvr and phr repair mechanisms and thus must utilize the SOS response thereby incurring more mutations. Various groups exposed their bacteria to UV light for a period of assigned time, photographed the cells, and then made dilutions accordingly to the exposure time so the cells could be practically counted and then backcalculated based on the dilution used. Viable cells were then calculated using the colored cells in the class spreadsheet. Time 0 was the initial amount of cells before UV exposure, thus, viable cells at each time point was “Cells at time x/ cells at time 0”. The initial amount of cells at time 0 was 126500000.
3.
After calculating the fraction of viable cells as mentioned above, Excel was used to visually graph the effect of of UV exposure of the given E. coli cells. This is considered a “survival’ or “killing” curve. As expected, the fraction of viable cells decreases as the time of exposure to UV light increases. The log of viable cells was used for this graph.
4.
Phase contrast microscopy at 40x magnification was used to observe the effects of UV exposure on the length of the E. coli cells. As mentioned earlier, the the E. coli W3110 is forced to used the SOS response to repair damage to light. The SOS response is induced by the single stranded DNA produced during faulty DNA elongation due to the dimers caused by UV damage which prevents proper DNA elongation. Once DNA polymerase is blocked from continuing DNA elongation, Rec A then cleaves the repressor which then indcues the operon and thus genes the halt cell division. It is this halting of cell division by the SOS response that accounts for the general trend of increasing bacteria length based on UV exposure time. Some photos have relatively poor quality making lenth hard to discern, nonetheless, the increase in length can still be generally observed.
5.
MAC agar was used to plate the dilutions of UV exposed E. coli in order to investigate whether the given sample was able to ferment lactose and to also investigate whether or not it was possibly a lac minus mutagen based. On MAC agar lactose fermenters will cause the agar to appear pink due to a decreased pH and non-fermenters will cause the agar to be yellow in color due to a basic pH due to breakdown of proteins. The E. coli sample used by my group was a lactose fermenter and did appear to be a possible lactose minus mutant as indicated by the coloring around the colonies indicated by the bright blue arrow.
6.
The given E. coli was then further evaluated for its ability to produce beta-galactosidase, an enzyme used to breakdown lactose. The bacteria were put into a TYE broth and incubated in 20-30 minute intervals and the absorbance values which correspond to the cells present in the mixture were recorded. Once the OD reached 0.5, IPTG was used to force the sustained induction of beta-galactosidase. Our group was using a possible mutant and could ferment lactose, thus it should show production of beta-galactosidase, however this was further evaluated using Miller Units.
7. The semilog above, of OD600 and time, can easily and quickly be used to find the generation, or doubling time, of the given bacterium. The time difference between the points at which and OD value doubles is the generation time as the OD value corresponds the the amount of cells in a mixture.
8.
After using IPTG to induce beta-galactosidase production, the culture was diluted accordingly so that cells could be counted, back calculated, and then used to calculate the fraction of viable cells.
9.
The colonies counted the day after beta-galactosidase production was induced with IPTG were used to calculate the fraction of viable cells. This was done done first by finding the initial amount of cells. It is known that an OD of 0.8 = 5 * 10^ cells/mL for E. coli W3110. Induction occurred for us at 70 minutes at an OD value of .405. Thus, the amount of initial cells can be found by (.405/.8)5 * 10^ cells/mL, this means that the initial amount of cells when induction occurred was 2.53125*10^8. Using the colonies counted following induction the amount of cells can be calculated using the dilution factor and amount counted, 10^5*10*280, thus 2.8*10^8. The fraction of viable cells at time of induction was then calculated by dividing 2.53125*10^8/2.8*10^8, thus 1.10167 is the fraction of viable cells at time of induction.
10.
Miller units were used to evaluate if the given bacteria produces beta-galactosidase as Miller units correspond to ONPG and B-galactosidase activity. Miller units are calculated via the formula where “t” is time and “v” is volume. In our case, induction started at 70 minutes so that was used as t(0) and the 20μL and 50μL were calculated separately for each time point. We saw beta-galactosidase activity that increased overtime as seen in by observing the Miller Units. This further confirms the assumption that the E. coli W3110 we used was lac mutant that can ferment lactose.
11.
In order to find the likely type of mutation that the mutant possess, the averages of the Miller Units of positive control lactose fermenters, negative control lactose non-fermenters, and the mutants were graphed in Excel using the class data.
One group did not list the identity of their bacteria and one group had listed invalided as their values, so those two groups data were thrown out. As seen above in the graph the positive control shows moderate beta-galactosidase activity and the negative control shows no activity as expected. Looking at the mutant, a large positive trend is seen in Miller Units overtime, more so than the positive control. Thus, the assumption can be confidently made the the mutant can in fact ferment lactose, which aligns with the MAC photos seen earlier. This also means that the mutation itself is likely not in the Lac-Z gene nor the promoter as both would prevent transcription and thereby production of beta-galactosidase. The gene that possess a mutation is likely a missense mutation in the Lac-Y gene or another gene that would still allow for transcription and thus production of beta-galactosidase as seen
above.
12. Conclusion: Overall, the lab was carried out successfully. The lab gave a thorough understanding of how serial deletions can be used effectively in evaluating the effect of cell survival based on something like UV exposure or to calculate the amount of viable cells before induction with IPTG. Graphs were created and showed the expected results in regards to survival after UV exposure, as well as growth with incubation time and IPTG induction of b-glucosidase. The mutant was confidently assumed to be a lac (-) with the ability to ferment glucose which then allowed for the deduction of what gene the mutation would likely be in. No major errors were observed in the lab.
and the lac operon was induced, the absorbance values were then used to plot growth vs. time. The amount of beta-galactosidase production of the potential mutant was then evaluated by calculating miller units which corresponds to b galactosidase production. The clas data miller units were then aggregated and evaluated to discover the identity of the mutant.
2.
UV Exposure and Colonies Observed
Fraction of Viable Cells and Time
E. coli W3110 was used as the bacteria for the UV exposure portion of the experiment as it has both mutations in the uvr and phr repair mechanisms and thus must utilize the SOS response thereby incurring more mutations. Various groups exposed their bacteria to UV light for a period of assigned time, photographed the cells, and then made dilutions accordingly to the exposure time so the cells could be practically counted and then backcalculated based on the dilution used. Viable cells were then calculated using the colored cells in the class spreadsheet. Time 0 was the initial amount of cells before UV exposure, thus, viable cells at each time point was “Cells at time x/ cells at time 0”. The initial amount of cells at time 0 was 126500000.
3.
After calculating the fraction of viable cells as mentioned above, Excel was used to visually graph the effect of of UV exposure of the given E. coli cells. This is considered a “survival’ or “killing” curve. As expected, the fraction of viable cells decreases as the time of exposure to UV light increases. The log of viable cells was used for this graph.
4.
Phase contrast microscopy at 40x magnification was used to observe the effects of UV exposure on the length of the E. coli cells. As mentioned earlier, the the E. coli W3110 is forced to used the SOS response to repair damage to light. The SOS response is induced by the single stranded DNA produced during faulty DNA elongation due to the dimers caused by UV damage which prevents proper DNA elongation. Once DNA polymerase is blocked from continuing DNA elongation, Rec A then cleaves the repressor which then indcues the operon and thus genes the halt cell division. It is this halting of cell division by the SOS response that accounts for the general trend of increasing bacteria length based on UV exposure time. Some photos have relatively poor quality making lenth hard to discern, nonetheless, the increase in length can still be generally observed.
5.
MAC agar was used to plate the dilutions of UV exposed E. coli in order to investigate whether the given sample was able to ferment lactose and to also investigate whether or not it was possibly a lac minus mutagen based. On MAC agar lactose fermenters will cause the agar to appear pink due to a decreased pH and non-fermenters will cause the agar to be yellow in color due to a basic pH due to breakdown of proteins. The E. coli sample used by my group was a lactose fermenter and did appear to be a possible lactose minus mutant as indicated by the coloring around the colonies indicated by the bright blue arrow.
6.
The given E. coli was then further evaluated for its ability to produce beta-galactosidase, an enzyme used to breakdown lactose. The bacteria were put into a TYE broth and incubated in 20-30 minute intervals and the absorbance values which correspond to the cells present in the mixture were recorded. Once the OD reached 0.5, IPTG was used to force the sustained induction of beta-galactosidase. Our group was using a possible mutant and could ferment lactose, thus it should show production of beta-galactosidase, however this was further evaluated using Miller Units.
7. The semilog above, of OD600 and time, can easily and quickly be used to find the generation, or doubling time, of the given bacterium. The time difference between the points at which and OD value doubles is the generation time as the OD value corresponds the the amount of cells in a mixture.
8.
After using IPTG to induce beta-galactosidase production, the culture was diluted accordingly so that cells could be counted, back calculated, and then used to calculate the fraction of viable cells.
9.
The colonies counted the day after beta-galactosidase production was induced with IPTG were used to calculate the fraction of viable cells. This was done done first by finding the initial amount of cells. It is known that an OD of 0.8 = 5 * 10^ cells/mL for E. coli W3110. Induction occurred for us at 70 minutes at an OD value of .405. Thus, the amount of initial cells can be found by (.405/.8)5 * 10^ cells/mL, this means that the initial amount of cells when induction occurred was 2.53125*10^8. Using the colonies counted following induction the amount of cells can be calculated using the dilution factor and amount counted, 10^5*10*280, thus 2.8*10^8. The fraction of viable cells at time of induction was then calculated by dividing 2.53125*10^8/2.8*10^8, thus 1.10167 is the fraction of viable cells at time of induction.
10.
Miller units were used to evaluate if the given bacteria produces beta-galactosidase as Miller units correspond to ONPG and B-galactosidase activity. Miller units are calculated via the formula where “t” is time and “v” is volume. In our case, induction started at 70 minutes so that was used as t(0) and the 20μL and 50μL were calculated separately for each time point. We saw beta-galactosidase activity that increased overtime as seen in by observing the Miller Units. This further confirms the assumption that the E. coli W3110 we used was lac mutant that can ferment lactose.
11.
In order to find the likely type of mutation that the mutant possess, the averages of the Miller Units of positive control lactose fermenters, negative control lactose non-fermenters, and the mutants were graphed in Excel using the class data.
One group did not list the identity of their bacteria and one group had listed invalided as their values, so those two groups data were thrown out. As seen above in the graph the positive control shows moderate beta-galactosidase activity and the negative control shows no activity as expected. Looking at the mutant, a large positive trend is seen in Miller Units overtime, more so than the positive control. Thus, the assumption can be confidently made the the mutant can in fact ferment lactose, which aligns with the MAC photos seen earlier. This also means that the mutation itself is likely not in the Lac-Z gene nor the promoter as both would prevent transcription and thereby production of beta-galactosidase. The gene that possess a mutation is likely a missense mutation in the Lac-Y gene or another gene that would still allow for transcription and thus production of beta-galactosidase as seen
above.
12. Conclusion: Overall, the lab was carried out successfully. The lab gave a thorough understanding of how serial deletions can be used effectively in evaluating the effect of cell survival based on something like UV exposure or to calculate the amount of viable cells before induction with IPTG. Graphs were created and showed the expected results in regards to survival after UV exposure, as well as growth with incubation time and IPTG induction of b-glucosidase. The mutant was confidently assumed to be a lac (-) with the ability to ferment glucose which then allowed for the deduction of what gene the mutation would likely be in. No major errors were observed in the lab.