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Request for Designation as a Scientific Methodologies (SM) Course in Explorations Name______Pete Van Zandt_______________________________________________ Course number and title_____ BI 101: Explorations in Biology____________ Departmental endorsement______________________________________________ Has this course been submitted for any other Explorations designation? ___No___ If so, which one? ______________ Please list which of your course assignments or activities addresses each of the guidelines, state briefly how this is accomplished, and attach a syllabus or a preliminary redesign plan for the course. Criteria for a course in scientific methodologies include Clearly defines a problem/question and states an appropriate rationale for investigation o An excerpt from the relevant laboratory exercises, wherein students learn about investigating natural phenomena using experimentation, is in Appendix 1 below. Briefly, students are required to examine either the behavior of termites (Lab 1) or the genetic inheritance pattern in fruit flies (Labs 4 – 6). Develops a testable hypothesis o (see Appendix 1) For relevant labs, students generate hypotheses either on their own (Lab 1), or are guided through this process from a limited number of possibilities (Labs 4 – 6). Tests the hypothesis using a suitable design, effectively analyzes resulting data, and draws proper conclusions o (see Appendix 1) For relevant labs, students evaluate these hypotheses either by designing experiments (with the instructor’s help) to test the hypotheses they develop (Lab 1), or through established experimentation and data analysis (Labs 4 – 6). Communicates the findings in oral or written form o For both exercises, the students prepare a laboratory report of their findings. Return this form as one electronic file with a syllabus appended to [email protected] by 30 May 2011. (See Appendix 2) Appendix 1 – Selected laboratory exercises for BI 101 Lab 1: The Scientific Process with Termites Work in groups of 3-4. On your desk, you should have a Petri dish with 4-5 termites in it. Take out several different pens and draw circles (about 2-3 inches diameter) on the supplied paper. Using the paintbrushes, gently brush the termites out of the Petri dish into the center of one of the circles. Observe the termites for 5-10 minutes, and then answer the questions below: 1. Identify a behavior that you would like to investigate further 2. Develop a TESTABLE hypothesis to explain the behavior 3. Design a controlled experiment (using as many termites as you want) to test your hypothesis. When doing this, be sure to discuss and identify the following terms for your experiment (this will be based on your previous knowledge, so you’ll have to share ideas with each other). Experimental Design: • Null hypothesis • Independent variables • Dependent variables • Experimental treatment • Control treatment • Sample size 4. Now the hard part…Choose an ecological phenomenon that interests you and your group. How might you use the scientific method to investigate this phenomenon? Labs 4-6: Investigating Mendelian Genetics Using Drosophila Introduction Genetics is the study of two main subjects, heredity and variation. Heredity determines the similarities between individuals. Children resemble their parents, puppies resemble their parents, and corn plants look like their parent plants. Variation is the cause of differences observed between individuals. Siblings resemble one another but are not identical, they are still unique individuals. Genetics attempts to explain the basis for the similarities and differences observed in related individuals. Humans have been curious about how characteristics are passed on from one generation to the next for thousands of years and although they did not understand the basis of heredity they quickly learned to apply genetic principles in the domestication of plants and animals. Initially, the plants or animals having the most desirable characteristics were used to produce the next generation. As the scientific basis of heredity became known, specific genetic principles were applied to produce plants and animals for specific purposes and for particular environments. From the earliest attempts by humans to explain inheritance of traits, the prevalent theories to explain heredity centered on the concept of "blended inheritance." This concept held that there was a blending or mixing of characteristics from the two parents, which led to the production of offspring appearing intermediate between the parents. It is easy to understand how this idea came into being and gained acceptance by the scientific community even into the late 19th century. However, with critical, objective observation and reasoning you can readily see why this is not an acceptable explanation for inheritance of traits. Consider your own physical characteristics. Are they midway between those of your parents? Are there any traits such as height, eye color, hair color, skin color, nose length, etc., for which you are exactly midway between your parents? Are there any for which you are more extreme than your parents? Are you perhaps taller than both of your parents? Any examples where the offspring are more extreme than both parents should be impossible if blended inheritance is the correct model for heredity. The correct explanation of the mechanism of heredity came with the publishing of a paper by an Austrian monk, Gregor Mendel, in 1866. Based on his results from hybridization experiments with garden peas, Mendel proposed the idea of hereditary "factors" or units. These factors later came to be called genes. Mendel proposed that these units were inherited in equal numbers from each parent and these determined the observable characteristics of the hybrid. The traits themselves are not inherited, but the factors, the units, or particles that determine or control the observable characteristic are transmitted from parent to offspring. The particular combination of factors inherited from the two parents determines the characteristic(s) of the offspring. Mendel's experiments led him to propose several laws or principles on which heredity is based. These laws are the foundation of what is typically called Mendelian genetics. The term "Mendelian genetics" refers to the subset of genetics concerned with the transmission of characteristics from one generation to another. Its focus is on the organism. Molecular genetics is concerned with the function of the gene. Its focus is below the level of the organism. Mendel's first law is called the "Law of segregation of alleles." This law states that when an organism reproduces, the "factors" for a trait are segregated—i.e. they are separated into different gametes. Mendel was not aware of chromosomes or the process of meiosis and so he used the term "factor" for describing his concept of particulate inheritance. As we understand it today each individual in a typical diploid eukaryotic population has two nonidentical copies of each chromosome and each chromosome contains numerous genes. Therefore every individual has two copies (alleles) of each gene or factor. These alleles may be the same, or they may be different. When an individual forms gametes (sperm and eggs), each gamete (which is haploid) carries only one allele for each trait. When two gametes combine (fertilization) they produce a new individual (which is diploid) with two alleles for each trait. The relationship between the two alleles for a trait helps explain why one does not always observe a blending of traits in the offspring of a given mating. One allele may be dominant to the other and in such a case will be expressed in the individual even if only one copy of that allele is present. The other allele in such a case would be recessive and its expression is masked by the dominant allele. If an individual has both of the dominant or recessive alleles for a given trait then that individual is homozygous for that trait. If an individual has one dominant and one recessive allele for a given trait then that individual is heterozygous for that trait. Two parents may both express the dominant form of a trait but be heterozygotes. An individual's appearance is referred to as its phenotype but the individual's genetic make up is referred to as its genotype. Therefore, an individual who was heterozygous for albinism (albinism is inherited as a recessive trait; normal skin pigmentation is dominant), would have a phenotype described as normal pigmentation. Such an individual could be described as having a genotype of Aa, where A represents the dominant allele for normal pigmentation and a represents the recessive allele for albinism. If two such heterozygous individuals should marry and have offspring then they could produce offspring having any of three possible genotypes, AA, Aa, or aa, and two possible phenotypes, normal pigmentation or albinism. Either AA or Aa would produce a phenotype of normal pigmentation. Heterozygous individuals are often referred to as carriers because they have the recessive allele, but do not express it. Heredity is usually more complicated than the above example, which shows a pattern of simple, dominant inheritance. Some traits exhibit what is referred to as codominance, a pattern of inheritance in which both alleles are expressed in the heterozygous individual. In addition, multiple alleles, polygenic inheritance, gene interactions, pleiotropy, etc., all contribute to the complexity and difficulty of understanding the genetics of an organism. Mendel's second law is the "Law of independent assortment." This law states that genes for different characteristics are inherited independently of one another. That is, the distribution of the alleles controlling one characteristic to the gametes does not affect the distribution of the alleles controlling a different characteristic to the gametes. This law holds only if the genes controlling the traits are located on different chromosomes. Procedures This investigation of Mendelian genetics using Drosophila will require three weeks because of the life cycle of the fruit fly. Specific tasks must be performed each week in order to complete the study within this time frame. Drosophila was one of the first organisms to be used extensively in genetic breeding experiments. It has continued to play an important role in genetic studies, and today is certainly the best known and most widely used species for these investigations. Its short life cycle (approximately 2 weeks), production of large numbers of offspring, ease of growing in the laboratory, and the large variation in inherited characteristics, all make Drosophila an excellent research organism and an ideal organism for beginning genetic studies. You will use the following timetable for completing your genetic studies using Drosophila: First Lab Period 1. learn how to handle and anesthetize fruit flies 2. learn the life cycle of the fruit fly 3. observe and learn to distinguish between the different mutations that have been used in the crosses you will be investigating 4. learn how to distinguish the sexes of fruit flies 5. prepare media in culture tubes for growth of your flies Second Lab Period 1. remove adult fruit flies from the culture(s) you started in the first lab period Third Lab Period 1. score (record) the results of the experimental fruit fly cross(es) you started in the first lab period 2. analyze your results using a chi-square statistical test First Laboratory Period Before beginning genetic study of any organism it is useful to become familiar with its life cycle. You should spend the first part of today's laboratory becoming acquainted with the life cycle of Drosophila and learning to recognize specific mutant traits present in some of the stocks of flies. Drosophila is a holometabolous insect, i.e. it undergoes complete metamorphosis and thus passes through four distinct stages during its life cycle—egg, larva (maggot), pupa and adult. Obtain a culture tube containing wild type flies and you should be able to observe all 4 stages inside the tube. Some ten hours after the female emerges from the pupa case she is ready for mating. Following a brief courtship during which the male circles the female while vibrating his wings, the female spreads her wings laterally and insemination takes place. Once sperm are received from a male they are stored in seminal receptacles within the female and are then used to fertilize all of the eggs laid by the female during the course of her lifetime. Some two days after emergence the female begins oviposition. She will lay eggs for some 10 days and produce some 400-500 eggs during this time. EGG. The eggs are small (0.5 mm long), white and ovoid. They have thin filaments or stalks on the anterior end that project from the egg case. These stalks serve to prevent the egg from sinking and drowning in the softened medium and also aid the developing embryo in respiration. This stage lasts about 1 day and ends when a small larva crawls out. They can be observed with the use of a hand lens or dissecting microscope. LARVA. The larvae are white, segmented and worm-like. There are black, hook-like mouth parts in the narrow head. There are no eyes and no appendages. The larvae eat their way through their environment. What is the natural environment of the fruit fly larvae and why are these morphological specializations advantageous there? A larva passes through 3 larval stages called instars. At the end of each instar they must shed their exoskeleton or molt. The larval stages take about 4 days to complete. How do the larvae move without appendages? PUPA. At the end of the last larval stage (3rd instar), the larvae move to a slightly drier location, become immobile, and evert their anterior breathing spiracles. They form a pupal case or puparium, which is analagous to the cocoon of a moth. It is within the puparium that metamorphosis occurs. Metamorphosis involves reformation of almost all body structures. The larval tissues are internally digested and the adult organs develop, all within a protective case. Over the 4 days that this stage lasts, the puparium hardens and darkens to a brown color. ADULT. The adult is the reproductive and dispersal stage of Drosophila. As the adult emerges from the puparium it is soft, pale and wet. At first the fly is very long with unexpanded wings which appear small and black. Within a short time the wings expand and the body takes on the shape of the mature adult. Flies are light in color upon emergence but darken during the first few hours. Recently emerged adults are difficult to sex and score for mutations that depend on development of coloration. Mating of the newly emerged flies will begin some 8-10 hours after leaving the puparium. Note: See Appendix II, "Techniques for Working with Drosophila," for figures showing stages in the life cycle of Drosophila and the parts of adult flies. Differentiating Sexes and Mutants Obtain some anesthetized adult flies from your instructor. Use a dissecting microscope and the figures in Appendix II and in the laboratory to identify the following body parts: Head, eyes, thorax, wings and their veins, halteres, abdomen, six legs, bristles, and antennae. Distinguish between males and females using the following characteristics: Size – Females are usually larger than males. Shape – The abdomen of males is rounded, blunt and cylindrical. Females have an abdomen that curves to a point posteriorly. Color – Males have the dark bands of the last few segments fused. Females have less black pigment and have separate dark bands along the dorsal surface to very tip of the abdomen. Sex Combs – Only males have a sex-comb. This is a darkly bristled structure located on the anterior pair of legs of the male. It is in a position analogous to the lower leg and ankle region. This is the most reliable method for positive sex identification. Note that the wings of the wild type flies are longer and wider than the abdomen. Note also that the eyes of wild type flies are red. The mutant traits that will be used in this investigation are white eyes, sepia eyes (brown), vestigial wings (wings are shrunken and nonfunctional). (Note: Flies that are newly emerged from the pupa case have unexpanded wings that are wet and folded and may appear vestigial. However, these wings are still longer than the fly's abdomen.) Examine flies that show each of the mutant traits listed above. You will not be told the genotypes of the flies that you receive for your experimental cross(es). Anesthesia – Flies will be anesthetized with triethylamine (FLYNAP). The goal is to inactivate the flies but not kill them. Your instructor will demonstrate several techniques for anesthetizing the flies. For written instructions on anesthetizing flies see Appendix II, "Techniques for Working with Drosophila," in this lab manual. Preparation of the Experimental Crosses One normally begins with homozygous individuals when conducting an experimental cross. One of the parents is homozygous for one allele while the other parent is homozygous for an alternative allele at the same genetic locus. Usually one parent is wild type for the trait under investigation while the other parent shows the mutant phenotype for the trait under study. These pure breeding flies are referred to as the parental generation and are symbolized by using P1. The offspring produced by the P1 cross are called the first filial generation and are symbolized by using F1. If the F1 flies are allowed to interbreed they produce a second filial generation and are symbolized by using F2. By analyzing the proportions of wild type and mutated flies in the F2 generation you can determine the mode of inheritance of the mutation under study and ascertain the genetic make-up of the flies in the F1 and P1 generations. Before beginning your crosses be certain that you know where the incubator is located that will house your flies while they are growing and reproducing. The incubator will keep the flies near a constant temperature of 25o C. Higher temperatures increase the risk of mortality of the flies and lower temperatures lengthen the time need to complete the life cycle. Media Preparation – Obtain clean plastic culture vials, one for each cross you will be conducting. Use the dehydrated media purchased from Carolina Biological Supply Company to prepare the growth media for your flies. Following the example of your instructor add one capful of dry medium (food flakes) to a clean tube. Add one capful of distilled water to the culture tube containing the dry medium. Quickly swirl the contents to mix the media and water. Set the tube on the counter to allow the medium to solidify. (This takes about 30 seconds). Add a few grains of yeast to each tube of medium you prepare; careful, not too much. Immediately place a clean foam plug in the tube to prevent any flies from getting into your culture tube. What is the function of the yeast and why is so little added? Placing Flies in the Tube of Culture Medium – Place 10-20 flies in the tube of culture medium you just prepared from the appropriate stock tube provided by your instructor. The flies you will be adding to start your culture are F1 flies. The P1 cross was performed for you several weeks earlier to provide you with enough time to complete your investigation. Use the following protocol in transferring flies, or first anesthetize them, see Appendix II, "Techniques for Working with Drosophila," and then transfer the flies to the culture tube. 1. Label your culture tube with your name and that of your lab partner. Indicate the letter of the cross (this will be on the stock tube from which you obtain your flies), on your label. Put the date and the lab section you are in on the label as well. 2. Remove the foam plug from the culture tube containing the freshly prepared medium and invert the tube. 3. Tap the stock tube (containing the adult F1 flies) on the table top to knock the flies off of the foam plug and toward the bottom of the tube. 4. Swiftly remove the foam plug from the stock tube and place your inverted new culture tube over the stock tube. 5. Allow the flies in the stock tube to enter the new culture tube. 6. After about 10-20 flies have entered the new culture tube, remove it from the stock tube and place foam plugs back into each of the culture tubes. Always plug the bottom tube first and plug the inverted tube before turning it right side up. Mentally rehearse the steps you are going to follow before you actually start the procedure. Get your lab partner to assist you in this process. 7. If too many flies climb up into the inverted tube, tap both tubes as a unit to knock most of the flies into the bottom culture tube and start over. 8. Check to make sure that your new tube is labeled correctly. Place it in the incubator when you are finished. If you decide to anesthetize the flies before placing them in your new culture tube then it is crucial that you do not expose the flies to the FLYNAP too long since this may kill them or reduce their fecundity. You must type (i.e. observe the phenotype) your F1 flies either before or after you have started your cross. You start the cross of your F1 flies when you place them in your new culture tube. To observe the phenotype of the flies it is necessary to anesthetize them and observe them under a dissecting microscope. You know that both of their parents were from different pure-breeding stocks (homozygotes). One of the parents in all of the P1 crosses was wild type while the other parent had a mutant phenotype. Therefore, what do you know about the genotype of these F1 flies? Enter your observations on the F1 phenotypes on the data sheets provided in this exercise. In addition, fill in the other information requested on this sheet and retain it. You will need this information as you complete your investigation and collect your data for analysis. Second Laboratory Period Removing F1 Adult Flies – During the past week the F1 generation of flies have been actively laying eggs in the culture tubes you started last week. If you look carefully in the culture vials you will note the presence of eggs, larvae and pupae which represent the F2 generation of flies. If you do not see evidence of living larvae or pupae in your culture vial, or if there is evidence of a fungal infection, then show the culture vial to your instructor to obtain advice on how to proceed. In order to prevent confusion of the F1 adults with the F2 flies that will begin to emerge in a few days it is important that ALL of the F1 adults be removed in today's lab. Following the procedures outlined last week, (see Appendix II to refresh your memory), anesthetize the flies in your culture vial and place them in one of the fly morgues present in the laboratory. The morgue contains a detergent solution, which will cause the flies to sink to the bottom of the fluid and drown. Try to prevent any of your flies from escaping into the laboratory. After you have removed all of your adult F1 flies return your culture vials to the incubator to permit the continued growth and development of the F2 generation. Third Laboratory Period Making Hypotheses and Predictions – This week you will determine if the alleles for the mutations you are studying are inherited as dominants, codominants or recessives, and if they are X-linked or autosomal. Use the space provided on the next page to write out your hypotheses and the predictions of the ratios you would expect among the F 2 offspring for each of your crosses. (Note: Make preliminary observations of your F2 flies to determine if you are dealing with a monohybrid, dihybrid, or sex-linked cross, and if the mutation is inherited as a dominant or recessive before proceeding to generate appropriate hypotheses and predictions for each of the crosses.) For example, if you find that Cross #1 has only two phenotypes and the mutant allele is recessive to the wild type allele, and they are distributed among both sexes in approximately equal numbers then your hypothesis might look like the following: Data Collection—Counting F2 Offspring – Obtain your culture vials that contain the genetic crosses you started two weeks ago from the incubator. These vials hold the F 2 generation of flies for each of the crosses you started at that time. Anesthetize the flies in one of these vials with FLYNAP, following the procedures you have used previously. At this point in time it is not necessary to be concerned about anesthetizing them for too long a period, since these flies will all be placed in the morgue after you finish your counts. It is important to first determine how many phenotypes are present among the F2 flies and if there are any differences between males and females with regard to the presence of a particular phenotype. None of your crosses will have more than four different phenotypes present. Separate the different phenotypes into groups. You need to count a minimum of 100 total flies, so if you have less than this number you will have to return in a few days and repeat this procedure of anesthetizing and counting the adult flies. Record your counts in Table 1 on the data sheets provided. Be careful to record your data on the proper sheet (i.e, cross 1 goes on the sheet for cross 1). Determine an approximate ratio of the phenotypes you observed. For each of your crosses, compare your observed approximate ratio with your hypotheses and predictions and determine which hypothesis most closely fits the data collected. Testing Data Against Predictions – Analyze your results statistically using the Chi-square Test (see below). This procedure allows you to compare your observed numbers of offspring with your predicted numbers to determine if they are statistically different from one another. Can you accept your hypothesis or must you reject it? If you must reject your hypothesis, it may be useful to test a different hypothesis using the Chi-square analysis. Once you have accepted one of your hypotheses, deduce what the genotypes and phenotypes of the parental (P 1), and F1 generations must have been and complete the information on your Drosophila Data Sheets. After finishing analysis of your fly crosses turn in your completed data sheets and the Chisquare analyses that you performed on your data. Use the sheet entitled "Chi-Square Calculations" to show your Chi-square analysis. Each group should complete a total of 4 Chi square analyses, including one for their own data and one for the pooled lab data for both Cross 1 and Cross 2. DROSOPHILA RESULTS CROSS # 1 As you determine the phenotypes and genotypes, fill in the blanks: P1 Phenotype: (female)__________________ X (male)_____________________ P1 Genotype: (female)__________________ X (male)_____________________ F1 Phenotype: (female)__________________ X (male)_____________________ F1 Genotype: (female)__________________ X (male)_____________________ Date F1 cross started ___________ Date F1 flies removed ___________ Date F2 flies counted ___________ Table 1. The Phenotypes and Numbers of F2 Progeny Phenotypes Your observed Your observed Lab observed Lab observed totals ratio totals ratio Males (white eyes) Males (wild type) Females (white eyes) Females (wild type) TOTALS ------------------- ------------------ DISCUSSION QUESTIONS FOR CROSS 1 The possible inheritance patterns for Cross 1 are: (1) (2) (3) (4) Autosomal recessive (normal Mendelian inheritance) Autosomal dominant Sex-linked recessive Sex-linked dominant 1. The allele for white eyes in flies is recessive. Is it possible to tell that based on your observed ratios? Why or why not? 2. What patterns do you see among the different phenotypes? 3. What ratios of F2 flies do you expect if a trait is autosomal recessive? Below, use punnett squares to determine generate hypotheses about expected ratios of F2 flies, starting with the P generation. Assume individuals in the P-generation are homozygous. Hint: use R = allele for red eyes, r = allele for white eyes Hypothesis 1: Autosomal Recessive P generation and F1 progeny F1 generation and F2 progeny Parental cross_________x__________ F1 cross_________x__________ F1 progeny genotype __________ EXPECTED RATIO OF F2 GENERATION: Red-eyed flies: White-eyed flies ____________:____________ 4. What ratios of F2 flies do you expect if a trait is sex-linked recessive? Hint: use punnett squares as before (in this case, you will have two possible crosses, with the P generation either a cross between red-eyed females x white-eyed males or between white-eyed females x redeyed males- Do punnett squares for each). Assume individuals in the P-generation are homozygous. Hint: for sex linked traits, you must use XX for females and XY for males (attach appropriate alleles, R and r, to appropriate chromosomes) Hypothesis 2: Sex-linked Recessive (P = red-eyed females x white-eyed males) P generation and F1 progeny F1 generation and F2 progeny Parental cross_________x__________ F1 cross_________x__________ F1 progeny genotype __________ EXPECTED RATIO OF F2 GENERATION: Male white eye: male wild type: female white eye: female wild type ____________:____________:_______________:_____________ Hypothesis 3: Sex-linked Recessive (P = white-eyed females x red-eyed males) P generation and F1 progeny F1 generation and F2 progeny Parental cross_________x__________ F1 cross_________x__________ F1 progeny genotype __________ EXPECTED RATIO OF F2 GENERATION: Male white eye: male wild type: female white eye: female wild type ____________:____________:_______________:_____________ 5. Before running your Chi-Square test, what do you think is the inheritance pattern for Cross 1? 6. Conduct Chi-square tests of the hypothesis that you think is the most likely of these choices (your answer to question 5). Show your calculations below and use the attached Chi-Square calculation pages. 6a. Based on the expected ratios of your hypothesis, what are the expected numbers of flies per phenotypic category (USE THE SAME EXPECTED RATIOS for your group data, and for the total lab data)? Group Lab 6b. Conduct Chi-Square tests for your group data and for the totals for the lab on attached calculation sheets. 7. Chi-Square Results: Group: 2 = _____________________ ___________________ df = ___________ P-value = Are your observed results (from your group) consistent with expected values based on your hypothesis? Lab: 2 = _____________________ ___________________ df = ___________ P-value = Are the observed results from the entire lab consistent with expected values based on your hypothesis? 8. If there is a difference between the overall results from your group and the entire lab, what might be the reason for the difference? DROSOPHILA RESULTS CROSS # 2 As you determine the phenotypes and genotypes, fill in the blanks: P1 Phenotype: (female)__________________ X (male)_____________________ P1 Genotype: (female)__________________ X (male)_____________________ F1 Phenotype: (female)__________________ X (male)_____________________ F1 Genotype: (female)__________________ X (male)_____________________ Date F1 cross started ___________ Date F1 flies removed ___________ Date F2 flies counted ___________ Table 2. The Phenotypes and Numbers of F2 Progeny (ignore sex) Phenotypes Your observed totals Your observed ratio Lab observed totals Lab observed ratio wild type eyes, wild type wings sepia eyes, wild type wings wild type eyes, vestigial wings sepia eyes, vestigial wings TOTALS ------------------- ------------------ DISCUSSION QUESTIONS FOR CROSS 2 1. Based on your observed ratios, are the traits for sepia eyes and vestigial wings recessive or dominant? Explain. 2. What patterns do you see among the different phenotypes? 3. What ratios of F2 flies do you expect if the traits are autosomal recessive? Hint: use punnett squares or probability calculations to determine the expected ratios of F2 flies, starting with the P generation. Assume individuals in the P-generation are homozygous. Hint: Use R = allele for red eyes, r = allele for sepia eyes, W = allele for normal wings, w = allele for vestigial wings, remember that individuals have two alleles for each trait, and that gametes have one allele for each trait Both Traits Autosomal Recessive P generation and F1 progeny progeny Cross ____________x______________ ____________x______________ F1 generation and F2 Cross RATIO OF F2 GENERATION: (red eye, norm wing):(se eye, norm wing):(red eye, vg wing):(se eye, vg wing) _________________:________________:______________:______________ 4. Conduct Chi-square tests of the hypothesis that the traits are both autosomal recessive. Show your calculations below and on the attached Chi-Square calculation pages. 4a. Based on the expected ratios of your hypothesis, what are the expected numbers of flies per phenotypic category (USE THE SAME EXPECTED RATIOS for your group data, and for the total lab data)? Group Lab 4b. Conduct Chi-Square tests for your group data and for the totals for the lab on attached calculation sheets. 7. Chi-Square Results: Group: 2 = _____________________ ___________________ df = ___________ P-value = Are your observed results (from your group) consistent with expected values based on your hypothesis? Lab: 2 = _____________________ ___________________ df = ___________ P-value = Are the observed results from the entire lab consistent with expected values based on your hypothesis? 8. If there is a difference between the overall results from your group and the entire lab, what might be the reason for the difference? Conducting a Chi-square Test To test whether your observed ratios match a particular expected ratio, you will use a statistical procedure called a Chi-square test.. This procedure will help you determine which hypotheses about the inheritance pattern is correct. In science, it is not sufficient to say that something "looks close enough for me," or "that seem reasonable." In most experiments, data will not be clear-cut and you will need to conduct some type of statistical analysis to compare your results with your predictions in an objective way. The best statistical test to use for analysis of genetic inheritance patterns is the Chi-square test. Chi-square Analysis – The Chi-square test is useful for comparing observed numbers in different categories with predicted values. The Chi-square test tells you the probability that the difference in the results you observed in an experiment versus what you would expect or predict could be due to chance events alone. Observed numbers that are close to predicted values will give you a high probability that the slight difference was due to random events and you can have confidence that2your hypothesis that led to those predictions is a reasonable one. = The Chi-square formula is shown below: (O-E)2 E O = number observed E = number expected = summation of all categories Example Suppose that you count the number of F2 fruit flies and find a total of 80 flies. Of these 80 flies, 55 are red-eyed (wild type) and 25 are white-eyed. You might be expecting a 3:1 ratio if red is dominant to white and these genes have a straight-forward inheritance pattern. Your expected frequencies for 80 flies would then be 60 red-eyed and 20 white-eyed. How close are the observed values to the expected values? Use the Chi-square test to determine if the difference you observed has a reasonable probability of being due to chance alone or if the hypothesis should be rejected as unreasonable to account for the results obtained. Using the numbers given the calculations would look like the following: 2 = (25 – 20)2 + 20 (55 – 60)2 60 The reason there are two sets of numbers above is that there are two classes of offspring observed, white and red. The χ2 value is obtained by adding the results from these two calculations together, i.e. you obtain the summation. The results of the calculations: χ2 = 1.25 + 0.42 χ 2 = 1.67 Each Chi-square value has a certain probability of occurrence, depending on the number of categories or classes. This is quantified as the degrees of freedom, which is equal to the number of classes or categories – 1. The above example has the smallest possible number of degrees of freedom, 2 – 1 = 1. Statisticians have developed probability tables that permit you to determine the probability of obtaining a χ 2 value like yours using the appropriate degree of freedom. Use one of the Chi-square probability tables provided for you in the laboratory to determine the p value (probability value) for the above χ 2. If the χ 2 value has a probability of less than 0.05 (for one degree of freedom the p value at 0.05 is 3.84) then it indicates that the results obtained are not likely the result of chance events. This means that if the hypothesis is correct and if the experiment was repeated 100 times you would obtain a value greater than 3.84 only 5 times or 5% of the time. Since the χ 2 value of 1.67 for the above example is less than the χ 2 value at the p = 0.05 level you would say that the difference between the observed and expected values is not significant at the 0.05 level and you would accept the hypothesis that gave you the predicted (expected) values. Because you do not know the exact p-value that corresponds to your χ 2 value, you can present p as (0.25>p>0.10). If the p-value corresponding to your χ 2 value was less than 0.05, then your expected values and observed values are significantly different, suggesting that your hypothesis about the inheritance pattern should be rejected. CHI-SQUARE TABLE Degrees of freedom Probability of committing a Type I error 0.90 0.50 0.25 0.10 0.05 0.01 1 0.016 0.46 1.32 2.71 3.84 6.64 2 0.21 1.39 2.77 4.61 5.99 9.21 3 0.58 2.37 4.11 6.25 7.82 11.35 4 1.06 3.36 5.39 7.78 9.49 13.28 5 1.61 4.35 6.63 9.24 11.07 15.09 CHI-SQUARE CALCULATIONS Cross 1 group data: Phenotype category O E (observed # flies) (expected # flies) O-E (O-E)2 (O-E)2 E χ 2 = Sum: P-value Cross 1 lab data: Phenotype category O E (observed # flies) (expected # flies) O-E (O-E)2 (O-E)2 E χ 2 = Sum: P-value CHI-SQUARE CALCULATIONS Cross 2 group data: Phenotype category O E (observed # flies) (expected # flies) O-E (O-E)2 (O-E)2 E χ 2 = Sum: P-value Cross 2 lab data: Phenotype category O E (observed # flies) (expected # flies) O-E (O-E)2 (O-E)2 E χ 2 = Sum: P-value TECHNIQUES FOR WORKING WITH DROSOPHILA Anesthetizing Fruit Flies with Flynap The following procedures describe how to effectively anesthetize your fruit flies without killing them. In anesthetizing your flies care should be used to avoid releasing them into the room and to minimize their exposure to FlyNap. Keep the bottles of FlyNap tightly closed, keep the lid on your anesthetizer, and clean up spills. Method 1 1. Remove the large cap from the bottom of an anesthetizer. Place 3-4 drops of FlyNap on the foam plug. Do not add any if you can already smell FlyNap on the foam. Replace the large cap. Remove the small plug in the top of the anesthetizer. 2. Transfer flies from the culture vial containing food to the anesthetizer as follows: a. Tap the flies off the foam plug, quickly remove the foam plug and invert the bottle onto the small end of the anesthetizer. b. Hold the bottles together firmly to prevent escape of any of the flies. Holding the pair together as a unit, tap the flies into the anesthetizer. [Do not pound too hard which can cause food to go into the anesthetizer along with the flies] c. Quickly stopper the anesthetizer, remove the culture vial, stopper it and set it aside. All of the flies do not have to be cleared from a culture vial at once. 3. Watch the flies closely and open the anesthetizer when the flies are quiet. Place the anesthetized flies on a white card for examination. 4. If the flies do not stop moving then add additional FlyNap to the anesthetizer. 5. If you continue to have problems getting your flies anesthetized then ask your instructor for assistance. Method 2 1. Obtain a clean plastic culture vial and foam plug marked for FlyNap use. Remove the foam plug but keep it close at hand. Obtain some FlyNap and a black anesthetizing "wand." 2. Transfer flies from the culture vial containing food to the anesthetizing vial as follows: a. Tap the flies off the foam plug, quickly remove the foam plug and invert the empty anesthetizer onto the culture vial. b. Invert the entire assembly, so the culture vial is now on top. c. Hold the bottles together firmly and tap them as a unit to force the flies into the anesthetizer. [AVOID POUNDING] d. Quickly stopper the anesthetizer, remove the culture vial, stopper it and set it aside. All of the flies do not have to be cleared from a culture vial at once. 3. Dip the wand into the FlyNap and squeeze out the excess on the side of the bottle. Tap the flies down to the bottom of the anesthetizer, push the foam plug to one side with your finger, and insert the wand into the anesthetizing vial. The foam plug will keep the wand in place. 4. Watch the flies closely and open the anesthetizer when the flies are quiet. Place the anesthetized flies on a white card for examination. 5. IMPORTANT! The foam plug of the anesthetizer will get FlyNap on it. Do not use this plug in one of your culture vials since it may have sufficient FlyNap on it to kill your culture. Figures Showing Drosophila Eggs and Larvae Figures Showing External Structures of Drosophila Appendix 2: draft syllabus for BI 101, Fall 2011 BI 101: Explorations in Biology-Fall 2011 Birmingham-Southern College Course Syllabus Class: TTh 8:00-9:20 am in SSC 134 Class Instructor: Office Hours: Peter Van Zandt, Ph.D. ([email protected]) SSC 246 - Monday & Thursday 1:00 – 3:00, and by appointment Lab sections and instructors (all labs meet in SSC 101) Lab 1 (T 12:30 – 3:20 pm): Van Zandt Lab 2 (W 2:00PM - 4:50 pm): Shew Lab 3 (Th 12:30 – 3:20 pm): Shew Required materials: 1. Biology (Volume 3 – Evolution and Ecology). 2008. P.H. Raven et al. McGraw Hill:Boston, MA. 2. Lab manual. 2007. M. Gibbons, S. Duncan, H.W. Shew, and P. Van Zandt, available from the BSC bookstore. 3. EcoBeaker manuals for a) Islands & Natural Selection, and b) Sickle-cell Alleles, both available from the BSC bookstore. Course Web Page: You should already have access to the Moodle page for this class. Look here daily for lecture outlines, reading guides, figures, important announcements, extra credit opportunities, lab assignments, data sets, etc. Course Description: A course for non-science majors designed to provide an understanding of selected fundamental biological principles and processes. This course does not count towards the biology or biologypsychology major. The class will consist of four modules, ranging from the more traditional organismal biology fields of Ecology and Evolution to the more applied areas of Ethnobotany and Forensic Science. Course Goals: The goals of this course are for you to: Understand and implement the scientific method Gain proficiency with interpreting graphs and figures Work with classmates to better understand course concepts Develop writing skills Participate in class discussions Develop critical thinking and problem solving skills Become better informed citizens Expectations: To achieve the goals described above, each student is expected to abide by the Birmingham-Southern College Honor Code (see below) and all other college policies read in advance, prepare for and actively participate in class activities be an active participant in your education – ask lots of questions complete the exams, quizzes, assignments, and in-class activities respect all peers and instructors involved with this course. Course Policies: 1. The Birmingham-Southern College Honor Code: All students in this course are expected to maintain academic integrity and uphold the Honor Code at all times. Specifically in this course, the following are considered violations of the Honor Code: collaborating on work assigned for individual completion; consulting or possessing work (except exams) of those who have previously completed this course; overstating your level of participation in a group assignment; using unauthorized resources in the completion of exams, quizzes, and assignments; plagiarism; turning in work that is not your own; lying, stealing, and lack of adherence to the instructions on any examination, quiz, assignment, or course policies listed below. You are expected sign an honor pledge on all work done for credit. Any violation of the honor code will be reported to the Honor Council and will result in a zero on that assignment. Penalties imposed by the Honor Council may be in addition to this academic penalty, and often include academic probation, suspension, or expulsion. 2. Attendance & Participation: Successful completion of this course requires active participation. Therefore, attendance in the classroom is strongly encouraged. You are expected to be on time and to stay for the duration of the class meetings. 3. Learning Accommodations: Under the directives and guidance of the Americans with Disabilities Act (ADA) and Rehabilitation Act of 1973, we are committed to providing appropriate accommodations to meet the learning needs of disabled students. If you believe that you qualify for learning accommodations based official documentation, please contact me, and appropriate learning accommodations in accordance with the recommendations can be arranged. It is critical that you contact me within the first week of the course so that I can make the appropriate arrangements. If you believe that you have a learning disability, but have not self-identified please contact the BSC Counseling and Health Services by calling x4717. 4. Communication: Office hours are Monday and Thursday 1-3 pm, but please feel free to set up an appointment if you have class during those hours. I will check and respond to student email and phone messages as soon as possible. An email class list will be created and used often to communicate important information about the class, so you are expected to check your BSC email frequently. 5. Cancellations & Time/Location Changes: If class or lab are cancelled or if there is a change in time or location of class for any reason, an email announcement will be sent and posted on Moodle and a sign posted on the classroom or lab door as soon as possible. In the event that class is cancelled, you will be expected to complete the scheduled reading. You will also be expected to complete assignments due for the cancelled class. Labs will be rescheduled if possible. 6. Course Work & Evaluation: Letter grades, as defined by the BSC 2006-2007 Catalog, will be assigned at the end of the course based on the number of possible points that you can earn where 93-100% = A, 90-92% = A-, 87-89% = B+, 83-86% = B, 80-82% = B-, 77-79% = C+, 73-76% = C, 70-72% = C-, 67-69% = D+, 60-66% = D, and <60% = F. Point distribution Exams Final Exam Quizzes Lab assignments Paper Title, summary, outline First draft Final draft Participation TOTAL 1170 400 (4 x 100 ea) 200 100 (20 x 5 ea) 220 (11 x 20 ea) 25 50 125 50 Exams: There will be four regular exams worth 100 pts each and one cumulative final exam worth 200 pts. Each of the 4 regular exams will be designed to emphasize the material in one module, but material may be integrated across the modules. Exams will include a combination of multiple choice, short answer, and essay questions relating to your reading or information that we covered in class. You may also be asked to recall and interpret figures from the text or from presentations, but much of the focus will be on critical thinking rather than just memorization. You must understand the concepts in order to apply them to new situations. If you know in advance that you’ll be absent during an exam I will arrange for an alternate test that will be entirely essay based. Missed exams cannot be made up. If an exam is missed due to extenuating circumstances, i.e., documented and Provost office-approved medical or family emergency, the points from that exam will be assigned equally to the other exams. Quizzes: There will be 20 unannounced quizzes in class or lab throughout the semester, to ensure that you show up on time, are completing your reading assignments, and are prepared for class. The quizzes should not be difficult, provided you have done the reading. The total quiz grade will be equivalent to one test grade. Missed quizzes cannot be made up. Lab assignments: See the lab manual for information on lab policies and assignments. Paper: Briefly, you will be required to submit a term paper on a topic from one of the four modules of this class. The paper topic need not correspond with one of our class topics, but you will have to have your topic approved by your lab instructor. There is more complete information in the lab manual. Class participation: Participation in every class meeting is important. To earn full credit, you should ask questions and contribute your background, understanding, and opinions to class discussions. Turnitin.com: You will be turning in your assignments through turnitin.com, which also has tips on avoiding plagiarism. We have begun to use the website Turnitin.com as a tool to educate our students about what plagiarism is and how to avoid it by citing sources correctly. Talk with me if you have any problems, questions, or concerns. TurnItIn information on plagiarism: http://www.turnitin.com/research_site/e_home.html. Study skills: I strongly encourage you to visit the Study Skills web site from BSC’s Counseling and Heath Services Website:http://www.bsc.edu/campus/counseling/index.htm. Another good resource at BSC is the Academic Resource Center (ARC - http://www.bsc.edu/academics/arc/index.htm), which provides you help with paper writing, study skills, and can even put you in touch with a peer tutor for your classes. Late Assignments: Late work will always be accepted and awarded some points. Work is considered late if not turned in when the assignment was due. If an acceptable excuse is provided, there will be no late penalty for late work. Otherwise, late work will lose 5% of its total possible point value every 24 hours starting at the time the assignment was due. However, the maximum penalty is 50% of the point value of the assignment to encourage you to turn in late assignments, no matter how late they may be. Class and Lab Schedule: Date Day Week Topic Readings/Assignments Labs 30-Aug Th 1 Course introduction none No labs this week 4-Sep T 2 Community ecology I Raven et al., Ch. 56 Lab 1: Library tour / Termites 6-Sep Th 2 Community ecology II Raven et al., Ch. 56 11-Sep T 3 Population Dynamics Raven et al., Ch. 55 13-Sep Th 3 Human Population growth & Impacts Raven et al., pp. 11611166; pp. 1227 -1234 18-Sep T 4 Conservation Biology Raven et al., Ch. 59 Lab 3: Stream Sampling 20-Sep Th 4 Exam I 25-Sep T 5 Darwin and Evolution Raven et al., Ch. 21 Lab 4: Fruit fly I 27-Sep Th 5 Genetics Raven et al., pp. 395403 2-Oct T 6 Natural Selection Raven et al., pp. 404412 4-Oct Th 6 Sexual Selection Raven et al., pp. 11331137 9- Oct T 7 Coevolution Raven et al., pp. 11751178 11- Oct Th 7 Speciation and macroevolution Raven et al., pp. 433452 16-Oct T 8 Exam II No labs this week 18-Oct Th 8 NO CLASS Fall Break 23-Oct T 9 Basic Botany Raven et al., pp. 581584; 594-602 25-Oct Th 9 Useful plant parts h.o. available on BB 30-Oct T 10 Agriculture h.o. available on BB Lab 2: Tree Diversity Lab 5: Fruit fly II/EcoBeaker on Islands & Natural Selection (due in next week’s lab) Lab 6: Fruit fly III/EcoBeaker Sickle-Cell Alleles (due in next week’s lab) Lab 7: Supermarket botany Lab 8: Ethnobotany hike at Ruffner Mtn. Nature Center 1-Nov Th 10 Medicinal plants h.o. available on BB 6-Nov T 11 Stimulants & Drugs h.o. available on BB Lab 9: Worms on Drugs 8-Nov Th 11 Exam III 13-Nov T 12 Crime Scene Investigation h.o. available on BB Lab 10: Forensic Palynology 15-Nov Th 12 DNA h.o. available on BB 20-Nov T 13 Fingerprints h.o. available on BB 22-Nov Th 13 NO CLASS 27-Nov T 14 Forensic Anthropology h.o. available on BB 29-Nov Th 14 Forensic Entomology h.o. available on BB 4-Dec T 15 Exam IV 12-Dec W FINAL EXAM (9-12) No labs this week Thanksgiving Break Lab 11: Fingerprinting No labs this week