Optimization of Genetic Biology Laboratories: DNA Repair Mechanisms URES Fall 2011 Summary- Stephanie Hintzen Biology labs

are an important part of the educational and learning process for many students. Labs allow for a hands on learning process where students can apply the skills they learn in courses rather than memorizing the materials. Students have the opportunity to develop the manual dexterity, communication and creative thinking skills they will need to make the future advances in one of the many fields of sciences but only if the lab runs smoothly. The DNA Repair Mechanisms lab is one of the labs in the General Genetics course at UMW. It helps bring the concepts of mutagenesis, cell culture, sterile technique and DNA repair, to name a few concepts, together for students in a tactile and physical way. In the process of the lab the students utilize techniques such as pipetting, cell counts, and statistical analysis as a way to determine the rate of repair in the cell cultures. By having the opportunity to interpret consistent and reliable data, the students would better be able to comprehend process of the DNA repair mechanisms. Reviewing and optimizing for this laboratory, would allow for that reliable data. During this experience I learned a lot about problem solving through experiments. A large part of the process was planning on how to reduce external factors and work towards the desired results. The four main changes I would recommend are to make the antibiotics and the NAA plates within two weeks of the lab as it seemed that the penicillin broke down somewhere before the 4 week mark. I would also recommend that the E.Coli that is used to make the bacterial culture should never be grown on an
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ampicillin plate as it appear that those bacteria are more likely to grow on the penicillin plates without the UV induced mutations. The third change I would recommend is that the bacterial cultures be prepared as close to the beginning of the lab as possible as there seemed to be a loss of growth when the undiluted MgSO4 bacterial suspension was saved for a week. I would also like to see students doing three plates of NA dilutions to better display the 10 folds dilutions. The final result of my URES experience was to incorporate my findings and suggestions into the current laboratory protocol. The final version of the laboratory is included below and changes are highlighted in yellow to emphasize the changes.

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EXERCISE 10 DNA REPAIR MECHANISMS All organisms have mechanisms by which they repair DNA damage. Mutations that alter the sequence of DNA are one type of DNA damage that occurs in the cells of organisms. Mutations can occur in the somatic cells of an organism and cause damage to cells and tissues within that individual, or they can occur in the gametes and be inherited by offspring. If the change occurs in the coding sequence of a protein, there are three possible consequences: The mutation can be neutral. In this case, the change in sequence either does not alter the amino acid sequence or the change in the amino acid sequence does not alter the function of the protein. The mutation can be detrimental. In this case, the change in sequence does alter the ability of the protein to function. This alteration usually results in a partial, or complete, loss-of-function for that protein. In other words, this type of mutation is the most harmful to an organism. In rare instances, a mutation can result in a beneficial change and the organism. The change in DNA sequence may cause a protein to work more efficiently or gain a positive function. In other words, this type of mutation may make an organism better able to survive under specific environmental conditions. The creation of a mutation can be categorized as either spontaneous or induced. Spontaneous mutations arise due to rare errors during DNA replication or repair whereas induced mutations arise from exposure to certain forms of radiation including UV light and ionizing radiation. Chemical mutagens also cause induced mutations. When mutations are induced, organisms activate a variety of mechanisms to repair the DNA. These mechanisms can be either error free, and return the DNA to its original state, or error-prone and maintain the mutations created. The purpose of this exercise is to: 1. Calculate and compare spontaneous mutation rate and UV induced mutation rate in Escherichia coli. 2. Determine the impact of photolyase repair on the induced mutation rate. The bacterium E. coli is an ideal model organism that we will be using for studying mutation rates. E. coli is a generally nonpathogenic gram negative bacterium that is naturally prevalent in the intestines of warm-blooded animals. (There are a few pathogenic toxin-producing strains of E. coli including the 0157:H7 strain). Since E. coli are haploid organisms, any mutation induced in an individual will be immediately
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expressed (no masking of phenotypes). Additionally, it is relatively easy to induce mutations in E. coli using UV light. Finally, E. coli reproduces by asexual fission, and thus all individuals arising from a single parent cell are expected to be genetically identical. In order to accomplish the first objective of this laboratory it is necessary to determine the spontaneous and the induced mutation rate for E. coli. Spontaneous mutations often arise as a result of errors in base pairing during DNA replication and they occur at a characteristic frequency for any given organism. The induced mutation rate will be determined for bacteria exposed to UV light. UV light is particularly effective at generating a specific precursor for mutation known as a pyrimidine dimer. The ring structures of two adjacent thymine bases in a polynucleotide chain will absorb UV light and form a cyclobutane ring which links carbons 5 and 6 of each pyrimidine ring to one another. Adjacent thymine and cytidine bases can also be photoactivated to form a 6-4 linkage between the two bases (Figure 1). If a pyrimidine dimer is left in place, the DNA replication machinery cannot replicate that region and the bacterium will die during cell division. In order to reduce the effects of pyrimidine dimers, E. coli have several repair mechanisms, some of which are able to reverse the dimer crosslink while others are more error-prone and create mutations. In order to accomplish the second objective, it is necessary to understand the types of DNA repair present in E. Coli. One type of error free repair is photoreactivation, or direct repair. Pyrimidine dimers can be recognized by DNA photolyase which binds to the photodimer and, in the presence of visible light, will split the dimer. All photolyases contain 2 chromophores. One is always FADH2; the other is either a folate or deazaflavin. All photolyases require light in the range 300-500 nm to become active. The E. coli enzyme requires light in the range of 365-400 nm. The experiment will demonstrate the impact of this repair mechanism on UV-exposed E. coli by exposing a subset of the UVexposed cells to visible light.

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Figure 1. The effects of UV radiation on adjacent pyrimidine bases, cyclobutane ring (a) and T(6-4)T linkage (b). Another error-free repair system that can recognize and remove pyrimidine dimers is nucleotide excision repair (NER). A series of enzymes first recognize the lesion. Next they make cuts in the DNA backbone of the strand containing the lesion, remove a segment of this strand on either side of the lesion and finally, synthesize a new replacement strand. This type of repair can occur both in the light and in the dark. An error-prone repair system that acts on pyrimidine dimers is translesion DNA synthesis. When DNA polymerase stalls at one of these dimers, it is replaced by an error-prone polymerase that is able to continue synthesis through the lesion by adding nucleotides across from the dimer. However, these nucleotides are not base-paired with those involved in the dimer and at least one of these nucleotides is usually not complementary to its partner in the dimer. This mechanism is known as a fail-safe mechanism in that it enables the completion of DNA replication but it is dependent on other systems to eventually break the pyrimidine dimer (such as either method discussed above). The changes put in place by the error-prone polymerase may lead to a change in the phenotype of the organism that can be either a positive mutation and lead to a new phenotype or a negative mutation and be lethal. This system is also active in both light and dark. Information check: Which of the above mentioned mechanisms of repair occur in the dark? Which occur in light? Do any of the repair mechanisms work only in light or only in dark conditions? In which condition (light and dark) will error prone repair dominate?

Although most mutations, either spontaneous or induced, are lethal to cells, sometimes a mutation can lead to a positive change that will enhance the cells ability to survive. In this exercise, you will assess the spontaneous and UV-induced mutation rate by a genetic selection for E.coli that have acquired a positive mutation that lead to a switch from penicillin sensitive (Ps) to penicillin resistant (Pr). Penicillin is able to inhibit cell growth by blocking cell wall synthesis. While all bacterial cells in the culture will be able to grow on nutrient agar (NA plates), bacterial cells possessing mutations that enable it to build a cell wall in the presence of penicillin will also form colonies on nutrient agar plates containing the antibiotic (NAA). In this experiment, bacteria will be cultured and treated so that you will be able to determine the mutation rate for spontaneous mutations, induced mutations with error-free
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repair systems activated, and induced mutation with error-prone repair system activated. In order to determine the mutation rate for each condition, you will determine how many colonies/mL are resistant to penicillin compared to the total number of colonies/mL in the original culture. To get countable colonies on the both NAA and NA plates, you will need to make serial dilutions prior to plating cells. NOTE: Perform all procedures in this lab using sterile techniques.

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Supplies for experiments For this laboratory, students will work in groups of 2 and be assigned one of the conditions described below for plating. Next lab period, each group will count the colonies on their plates and the class will share all data. Group A spontaneous mutation rate Label 5 tubes containing 990 l of nutrient broth Label 2 NAA plates NAA1 and NAA2 ** Label 2 NA plates NA1, NA2 and NA3 Group B UV induced, light treated Label 5 tubes containing 990 l of nutrient broth Label 1 tube containing 900 l of nutrient broth Label 2 NAA plates NAA3 and NAA4 ** Label 2 NA plates NA4, NA5 and NA6 Group C UV induced, dark treated Label 5 tubes containing 990 l of nutrient broth Label 1 tube containing 900 l of nutrient broth Label 2 NAA plates NAA5 and NAA6 ** Label 2 NA plates NA7, NA8 and NA9

Performed by instructor This procedure is outlined on the left panel of Figure 2 Antibiotic plates (NAA) are made within 2 weeks of lab. Grow a culture of E. coli in 400 ml of nutrient broth for 24 hours at 37oC with vigorous shaking, within a week of the lab. The bacteria must be from a new culture of E. coli that has not been grown on ampicillin. This will yield a culture with approximately 107-108 cells/ml. Centrifuge the culture at 4000 rpm for 10 minutes, and resuspend the bacteria in 70 ml of 0.1 M MgSO4. I. 1. Spontaneous Mutation Rate Transfer 35 ml of the cell suspension to a new flask, add 35 ml of nutrient broth and incubate for 30 minutes. After the incubation, aliquot 2 ml of the bacterial culture into a sterile test tube for each lab group (Flask 1).

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Performed by the student group A PERFORM EACH STEP USING STERILE TECHNIQUE This procedure is outlined on the top panel of Figure 2 2. Pipet 1.0 ml of the culture onto the NAA1 plate (no dilution) and 0.1mL of the culture onto the NAA2 plate (10-1 dilution) and spread the bacteria using the bacterial spreader. These plates represent the number of colonies in the original culture that have been mutated and have acquired penicillin resistance. Since this culture was not exposed to UV light, these colonies were formed by spontaneous mutation. Transfer 100 l of the bacterial culture to Dilution tube 1 and mix (10-2). Then transfer 10 l from Dilution 1 to Dilution 2 and mix (10-4). Do another serial dilution by transferring 10 l from Dilution 2 to Dilution 3 and mix (10-6). Do another serial dilution by transferring 10 l from Dilution 3 to Dilution 4 and mix (10-8). Pipet 0.1 ml from Dilution 3 onto the NA1 plate (10-7), 1.0 mL of Dilution 4 onto NA2 (10-8) and 0.1mL of Dilution 4 onto the NA3 plate (10-9) and gently spread the bacteria using the bacterial spreader. These plates represent the total number of colonies in the original culture. After the fluid has soaked into the plates, place them (inverted) in an incubator at 37oC for 24 hours. After 24 hours, your instructor will move the plates to 4C to save them for next week. Induced Mutation Rates

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I.

In this portion of the lab, you will determine if exposure to UV radiation increases the mutation rate using antibiotic resistance as the indicator. You will also study the effects of exposing UV treated E. coli to visible light on the mutation rate. Performed by instructor 1. Place 25 ml of undiluted MgSO4 bacterial suspension (from part I) in a sterile petri dish with a magnetic stir bar. Place the petri dish on the magnetic stirring plate 15 cm from the UV light. Turn the stirrer on low speed. Turn off room lights to limit photolyase activity. Remove the lid and expose the bacteria to the UV light (Labeled # 1- short wave) for 30 seconds. Add 25 ml of nutrient broth to the culture, mix, and immediately transfer 25 ml of the culture to each of two flasks. One flask is covered with aluminum foil. Incubate both flasks for 30-45 min. with vigorous shaking at 37oC. At the end of the incubation period, distribute 2 ml of each culture, light (group B) and dark (group C) exposed, to each lab
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group. Performed by the student group B PERFORM EACH STEP USING STERILE TECHNIQUE This procedure is outlined on the middle panel of Figure 2 2. Pipet 1.0 ml of the light culture onto the NAA3 plate (no dilution) and 0.1mL of the light culture onto NAA4 plate (10-1) and spread using a L-shaped glass rod. These plates represent the number of colonies in the original, treated, culture that have been mutated and have acquired penicillin resistance. Since this culture was treated with UV light and normal light, these colonies represent the induced mutations that were not repaired by nucleotide excision repair or photolyase. Make serial dilutions of the light culture by transferring 0.1 ml of the culture to Dilution tube 5, mix (10-2), and transfer 10 l from Dilution 5 to Dilution 6 (104 ). After mixing, transfer 10 l from Dilution 6 to Dilution 7 and mix (10-6). Do another serial dilution transferring 100 l ml from Dilution 7 to Dilution 8 and mix (10-7). Do another serial dilution transferring 100 l ml from Dilution 8 to Dilution 9 and mix (10-8). Pipet 1.0 mL from Dilution 8 to the NA4 plate (10-7) and 1.0 mL of Dilution 9 onto the NA5 plate (10-8) and .1 mL of Dilution 9 onto NA6 plate (10-9) and spread with the bacterial spreader. These plates represent the number of colonies in the original, treated, culture. Since this culture was treated with UV light and normal light, these colonies represent bacterial cells in the original culture that survived UV exposure. The difference between the number of colonies here and those from group A represents the number of cells that died because either the pyrimidine dimers formed were not repaired or the mutation acquired was a lethal mutation. After the fluid has soaked into the plates (at least 10 minutes) place them (inverted) in an incubator at 37oC for 24 hours. The instructor will remove the plates and keep them at 4C for the next lab period. Performed by student group C PERFORM EACH STEP USING STERILE TECHNIQUE This procedure is outlined on the bottom panel of Figure 2 2. Pipet 1.0 mL from the dark culture onto the NAA5 plate (no dilution) and 0.1 mL from the dark culture onto the NAA 6 plate (10-1) and spread using a bacterial spreader. These plates represent the number of colonies in the original, treated, culture that have been mutated and have acquired penicillin resistance. Since this culture was treated with UV light and but NOT normal light, these colonies
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represent the induced mutations that were not repaired by only nucleotide excision repair (not photolyase). 3. Make serial dilutions of the light culture by transferring 0.1 mL of the culture to Dilution tube 10, mix (10-2), and transfer 10 l from Dilution 10 to Dilution 11 (10-4). After mixing, transfer 10 l from Dilution 11 to Dilution 12 and mix (106 ). Do another serial dilution transferring 100 l ml from Dilution 12 to Dilution 13 and mix (10-7). Do another serial dilution transferring 100 l ml from Dilution 14 to Dilution 15 and mix (10-8) Pipet 1.0 mL from Dilution 14 to the NA6 plate (10-7) and 1.0 mL of Dilution 15 onto the NA6 plate (10-8) .1 mL of Dilution 15 onto NA6 plate (10-9) and spread with the bacterial spreader. These plates represent the number of colonies in the original, treated, culture. Since this culture was treated with UV light but NOT normal light, these colonies represent bacterial cells in the original culture that survived UV exposure. The number of colonies on these plates should be similar to the number that group B obtained. This is because both cultures came from the same original, UV treated sample. Exposure to normal light or dark should not have a noticeable effect of the survival rate. After the fluid has soaked into the plates (at least 10 minutes), place them (inverted) in an incubator at 37oC for 24 hours. The instructor will remove the plates and keep them at 4C for the next lab period.

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III. Calculation of Mutation rates (all groups) 1. During the next lab period, count the number of bacterial colonies on all of your groups' plates and record the data from your group on the board. Once all groups have entered their data, record in table 1 the average of the colonies counted for each plate (ONLY average the plates if the colony count is between 3 and 300. If no plates have a count between 3 and 300, ask your instructor for guidance). Use the dilution factor to calculate the number of colonies/mL in your original cultures and record your results in table 1. The dilution factor = the inverse of your dilution (for example, if your dilution was 10 -4, then your dilution factor is equal to 104 or 10,000.) If appropriate, average the number of colonies/mL from the plates that came from the same part of the experiment (For example, NAA1 and NAA2). It is appropriate to average IF the plates give a number of colonies/mL and the results are of the same magnitude. Ask your instructor if you are uncertain as to whether or not to average the numbers
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2.

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. Use the data to calculate the spontaneous and induced mutation rates as shown below:

a. Spontaneous mutation =

(average # of colonies NAA1 and NAA2) (average # of colonies NA1 and NA2)

b. Induced mutation frequency = (average # of colonies on NAA3 and NAA4) (visible light treated) (average # of colonies on NA3 and NA4)

c. Induced mutation frequency = (average # colonies on NAA5 and NAA6) (dark treated) (average # colonies on NA5 and NA6)

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Table 1. Bacterial Colony Counts Plate Number of Colonies on Plate Dilution Factor Number of Colonies/mL in original culture Average number of colonies/mL if appropriate

NAA 100l CONTROL NAA 1mL NA1 NA2 NA3 NAA 100l LIGHT NAA 1mL NA4 NA5 NA6 NAA 100l DARK NAA 1mL NA7 NA8 NA9

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POST LAB QUESTIONS This laboratory will be written up as an abstract. These questions are not an assignment, but they are designed to help guide you through the types of information that you should included in your abstract. 1. What was the hypothesis for this laboratory? (Be careful, the hypothesis is a general statement that does not to include E. coli or UV light.) 2. How did you test your hypothesis? (This is where you would briefly state the organism and procedure for mutation, and how you will measure results). 3. Rank your expectations from highest to lowest mutation rate for the spontaneous, UVinduced+dark, and UV-induced+light experiments. High ______________ _________________ __________________ Low Explain your rationale:

4. Do the experimental results correlate with your expectations? ______________ If no, do you think your expectations require reevaluation or do you think some other factor is involved?

What might have caused variation from expected in your experiments? 5. Why do you need to dilute the cultures so much prior to plating on NA media and not at all for plating onto NAA? 6. Why are the NA plates that have cultures treated with UV light diluted less (10-7 and 10-8) than those with cultures that were untreated (10-7 and 10-9)? 7. Do you know what the terms mutagenesis and mutagenize mean?

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RESULTS Table 2. Bacterial Colony Counts week 1 Plate Number of Colonies on Plate 0 0 560 49 11 8 93 307 47 12 7 54 250 15 2 Dilution Factor 101 No 108 109 1010 101 No 108 109 1010 101 No 108 109 1010 (0) = 0 (206) Number of Colonies/mL in original culture 0 0 5.6 x 1010 4.9 x 1010 1.1 x 1011 80 93 3.07 x 1010 4.7 x 1010 1.2 x 1011 70 54 2.5 x 1010 1.5 x 1010 2.0 x 1010 2 x 1010 62 3.9 x 1010 86.5 5.25 x 1010 Average number of colonies/mL if appropriate 0

NAA 100l CONTROL NAA 1mL NA1 NA2 NA3 NAA 100l LIGHT NAA 1mL NA4 NA5 NA6 NAA 100l DARK NAA 1mL NA7 NA8 NA9

a. Spontaneous mutation =

b. Induced mutation frequency = 2.2 x 10-9 (visible light treated) 3.1 x 10-9

c. Induced mutation frequency = (dark treated)

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Table 3. Bacterial Colony Counts week 2 Plate Number of Colonies on Plate 0 0 209 20 1 0 23 108 7 0 0 18 29 5 0 Dilution Factor 101 No 108 109 1010 101 No 108 109 1010 101 No 108 109 1010 (0) = 0 (76) Number of Colonies/mL in original culture 0 0 2.09 x 1010 2.0 x 1010 1.0 x 1010 0 23 1.08 x 1010 7.0 x 1010 0 0 18 2.9 x 109 5.0 x 109 0 3.95 x 109 9 2.63 x 1010 11.5 1.69 x 1010 Average number of colonies/mL if appropriate 0

NAA 100l CONTROL NAA 1mL NA1 NA2 NA3 NAA 100l LIGHT NAA 1mL NA4 NA5 NA6 NAA 100l DARK NAA 1mL NA7 NA8 NA9

a. Spontaneous mutation =

b. Induced mutation frequency = (23) = 4.3 x 10-10 (visible light treated) (2.63) c. Induced mutation frequency = (9) = 2.3 x 10-9 (dark treated) (3.95)
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