AP Biology Laboratory Report

AP Biology Laboratory Report Lab 1: Artificial Selection • Question: Can extreme selection change expression of a quantitative trait in one generation? • Learning Goals: - Investigate natural selection as a major mechanism of evolution - Apply mathematical methods to data from a real population to predict what will happen to the population in the future - Investigate how natural selection acts on phenotypic variations in populations Procedure: • • Part 1: Initial plant cultivation (p generation) o Variable selection o F1 generation cultivation o F1 generation analysis Part 2: F2 generation cultivation (optional) Parent Plant Generation Cultivation: - • Prepare growing containers and plant Wisconsin Fast Plants At 7-12 days students choose 1 variable trait for their focus - Trait should vary between plants and also between containers - Varies within a range o Height, leaf number, trichome number • • Compile a list of ALL possible traits your class identified Calculate appropriate descriptive stats for class data for the first generation - Mean, median, mode, range, std dev., etc. - Create a histogram that shows the frequency distribution of the trait you have selected *For the remainder of this section, I will be using trichome density as my variable, but understand that others are possible * • Directional selection - As a class choose a trait in which to move the variability in one direction or another - Find the top (or bottom) 10% of plants with the entire class’s population (e.g., out of a population of 150 plants, the 15 hairiest plants) and mark any that are in your bottle container. - Cut off the tops of the remaining plants (not the 10%) • • • • • • Construct a new histogram and calculate appropriate descriptive stats Isolate selected plants from the rest of the population On days 14-16, when several flowers are present, cross pollinate the selected plants with a single bee stick or pollinating device - WFP are not self pollinating - Collect and distribute pollen from every flower onto every other plant in the population Allow plants to complete their life cycle As seed pods form, be sure to limit each of the plants to 8 to 10 seedpods and several days later harvest the seed pods Cultivate Second Generation (F1) Of Plants - Two populations of seeds (selected plants and non-selected plants) Score plants on same cultivation days as you did generation 1 Analyze results - Are the two populations/generations before and after selection actually different? Are the means significantly different? Should you use median or mean as a measure of central tendencies at this point in the investigation Consider constructing a bar graph to compare the mean number of hairs per generation Include error bars, but first determine what is appropriate. What statistical test could you apply to help you define your confidence about whether these two populations are different? Compare the second population to the parent subpopulation of generation one. How do these two populations compare? How does this comparison differ from your other comparison? Lab 2: Mathematical Modeling: Hardy Weinberg • Question: How can mathematical models be used to investigate the relationship between allele frequencies in populations of organisms and evolutionary change? • Learning Goals: - Use a data set that reflects a change in the genetic makeup of a population over time and apply mathematical methods and conceptual understandings to investigate the cause(s) and effects of this change - Describe a model that represents evolution within a population - Evaluate data sets that illustrate evolution as an ongoing process Procedure: • • • Part 1: Setting up Mathematical Model Part 2: Testing/Analyzing Mathematical Model Part 3: Alter Model to Analyze Relationships Building a Mathematical Model 1. Formulate the question 2. Determine the basic ingredients 3. Qualitatively describe the biological system 4. Quantitatively describe the biological system 5. Analyze the equations 6. Perform checks and balances 7. Relate the results back to the equation Record thoughts, strategies and assumptions on modeling as you work through the next section 1. Formulate the question Think about a Mendelian trait such as cystic fibrosis • How do recessive alleles stay in the population? Why do they not gradually disappear? Think about a dominant Mendelian trait such as polydactyly (more than 5 fingers or toes) • It is a dominant trait, but it is not common in most humans…why? Do inheritance patterns or allele frequencies change in a population? 2. Determine the basic ingredients Simply the question with some basic assumptions (hint: remember rules for HWE) • For this model, lets assume: - All organisms are diploid (having a gene locus with two alleles, A and B) o We could use A and a as our alleles, but using A and B will be much much easier in the spreadsheet we are developing - Consider that the population has an infinite gene pool - Gametes for the next generation are selected at random 3. Describe the system - - Imagine the life cycle of our hypothetical organism All the gametes go into one infinite pool o All have an equal chance of taking part in fertilization and then formation of a zygote For now, all zygotes like to juveniles and then live to be adults No individuals enter or leave the population (no gene flow) No mutation 4. Quantitatively describe the system Our goal is to model how allele frequencies change through one life cycle of an imaginary population - Spreadsheets (such as Excel or Google Sheets) are valuable tools that can help us answer “what if” questions. o They can model randomness o Repeatedly make calculations based on another calculation Each part of the life cycle can be represented by a spreadsheet operation - • • Set allele frequencies - p = the frequency of the A allele - q = the frequency of the B allele • Use the random function (RAND) to generate a random number which will be compared to the allele frequency • Compare the random number to the allele frequency and assign the appropriate allele • We will introduce zygotes into the next group of cells - Zygote is a combination of two randomly selected gametes - You want to “concatenate” the values in the two cells • In the next columns, we will input our “if” statements, these cells are used for bookkeeping, that is keeping track of each zygotes genotype • You can now use this data to simulate subsequent generations • Testing Your Mathematical Model - What can you change in your model? If you change something, what does the change tell you about how alleles behave? - Do alleles behave the same way if you make a particular variable more extreme? Less extreme? Do alleles behave the same way no matter what the population size is? To answer this question, you can insert rows of data somewhere between the first row of data and the last row and then copy the formulas down to fill in the space. Try out different allele frequencies in the model Different cases: - Heterozygote advantage - Homozygous lethal - • • Lab 3: Comparing DNA Sequences to Understand Evolutionary Relationships with BLAST • Question: How can bioinformatics be used as a tool to determine evolutionary relationships and to better understand genetic disease? • • Learning Objectives: - Create cladograms that depict evolutionary relationships - Analyze biological data with a sophisticated bioinformatics online tool - Use cladograms and bioinformatics tools to ask other questions of your own and to test your ability to apply concept you know relating to genetics and evolution Pre lab: Cladogram Basics - Create a cladogram of the following plant groups Procedure: • • Part 1: Fossil Analysis Part 2: Using Blast • Part 3: Independent Design Procedure: - Hypothesize cladogram placement from fossil morphology Locate and DL gene files and upload to BLAST • • As you collect information from BLAST for each of the gene files, think about your original placement of the fossil species on the cladogram - The higher the score, the closer the alignment - The lower the e value, the closer the alignment - Sequences with e values less than 1e-04 (1 x 10-4) can be considered related with an error rate of less than 0.01%. Compare your cladogram with that of your classmates Lab 4: Diffusion and Osmosis • Question: What causes plants to wilt if they are not watered? • Learning Goals: – Investigate the relationship among surface area, volume and rate of diffusion - Design experiments to measure the rate of osmosis in a model system Investigate osmosis in plant cells Analyze the data collected in the experiments and make prediction about molecular movement through cellular membranes Procedure: • • • Part 1: Determine Relationship Between Surface Area, Cell Size and Rate Of Diffusion (Agar Cell “Races”) Part 2: Model Diffusion and Osmosis Using Dialysis Tubing Part 3: Observe Osmosis In Living Cells Part 1: Surface Area and Cell Size • • Think about specialized cell functions such as epithelial cells lining the intestine or plant root hairs… - What are the size/shape of these cells? You will be “designing” three different cell models and note trends between cell shape/size and diffusion rate - “cells” are soaked in pH-indicator dye phenolphthalein and placed in either 0.1M of HCL. 0.1M of NaOH or distilled vinegar - You will look for degradation of the dye in order to determine diffusion Part 2: Modeling Diffusion and Osmosis • You will create models of living cells using dialysis tubing - Dialysis tubing is made from material that is selectively permeable to water and some solutes - You will fill your model with different solutions and determine the rate of diffusion Part 3: Osmosis in Living Cells • • • • Observe a single leaf blade of Elodea under a light microscope Note major cellular structures Test solutions from part II Be sure to record your observations, calculations and procedures Lab 5: Photosynthesis • Question: What factors affect the rate of photosynthesis in living leaves? • Learning Goals: - Design and conduct an experiment to explore the effect of certain factors including different environmental variables on the rate of photosynthesis - Connect and apply concepts, including the relationship between cell structure and function (chloroplasts); strategies for capture, storage and use of free energy; diffusion of gases across cell membranes; and the physical laws pertaining to the properties and behaviors of gases Procedure: • • • Part 1: Leaf Disks Part 2: Design Your Own Experiment Part 3: Spectrophotometry (Optional?) Part 1: Leaf Disks - • Make leaf disks by hole punching spinach leaves Prepare bicarbonate solution to provide CO2 Set disks in light Photosynthesis releases O2 gas, which will form on disks Disks will gradually raise in solution Part 2: Design Your Own Experiment - Alter variables such as o Light color, intensity, bicarbonate concentration, etc. Part 3: Spectrophotometry • • Learning Goals: - Determine the absorbance/transmittance of various - Justify the use of primary pigments and secondary pigments used in photosynthesis Spectrophotometer determines the relative transmittance or absorbance of substances • Adjust wavelength and pigments in cuvettes Lab 6: Cellular Respiration • Question: What factors affect the rate of cellular respiration in multicellular organisms? • Learning Goals: - Learn how a respirometer system can be used to measure respiration rates in plant seeds or small invertebrates, such as insects or earthworms - Design and conduct an experiment to explore the effect of certain factors, including environmental variables, on the rate of cellular respiration - Connect and apply concepts, including the relationship between cell structure and function (mitochondria) Procedure: • • Part 1: Setting up your Microrespirometer Part 2: Independent Design Part 1: Setting up your Microrespirometer • Setting up Experimental Condition Setting up Control Condition Collecting Data Design and Conduct Independent Investigation Part 1: Setting up your Microrespirometer - • • A respirometer will measure the volume of gas present in a closed system In this lab you will create a simple respirometer using plastic tuberculin syringes without needles, capillary tubes, hot glue and nonabsorbent cotton. Typically, small insect or germinating seeds are used in order to perform cellular respiration KOH is placed on an absorbent cotton plug - KOH negates gaseous CO2 product CO2 + 2 KOH → K2CO3 (solid precipitate) + H2O • • • The respirometer tips are submerged in water As O2 in the respirometer is consumes, water is drawn into the tip This provides a direct measurement of oxygen consumption • 3 Conditions - Experimental o (all germinating peas ) - Experimental o (non germinating peas and glass beads) - Control (all glass beads) • The control group in this case accounts for several environmental factor that can alter the results - Bath temperature - Atmospheric pressure - Any bumps to the table, etc. If any water is up taken through the respirometer in the control setting (with nonrespiring glass beads) we can assume the volume is not due to cellular respiration • • Conduct Independent Investigation - Differences between species? Temperature effects? Germination time (several days vs. few days)? Spring seeds vs. summer seeds? Monocot vs. dicot? You could even try small invertebrates, but let’s try to be as ethical as possible • Analysis: - Be sure to record ALL data - Apply descriptive statistics - GRAPH Lab 7: Cell Division: Mitosis and Meiosis • Question How do eukaryotic cells divide to produce genetically identical cells or to produce gametes with half the normal DNA? • Learning Goals - Describe the events in the cell cycle and how these events are controlled - Explain how DNA is transmitted to the next generation via mitosis - Explain how DNA is transmitted to the next generation via meiosis followed by fertilization - Understand how meiosis and crossing over leads to genetic diversity necessary for evolution Procedure: • • • • • Part 1: Modeling Mitosis Part 2: Effects of Environment on Mitosis Part 3: Loss of Cell Cycle Control in Cancer Part 4: Modeling Meiosis Part 5: Meiosis and Crossing over in Sordaria Part 1: Modeling Mitosis - Review steps of the cell cycle and mitosis Create visual models using pop beads, clay, pipe cleaner chromosomes, etc. Be sure to identify crucial structures potential failures of the system Part 2: Effects of Environment of Mitosis - Task: solve fungal pathogen (lectin) effects on mitotic cells in plant root tips Prepare cell squash o Onion or garlic root tip squash • • Observe cells at a high magnification Within the field view identify and count cells in each phase • Collect class data in an observed values table • Compare to an expected values table • Calculate the Chi-Square Value Part 2 Analysis: • • • • • What was the importance of collecting class data? Was there a significant difference between groups? Did the fungal pathogen lectin increase the number of root tip cells in mitosis? Does an increased number of cells in mitosis mean that these cells are dividing faster than the cells in the roots with a lower number of cells in mitosis? What other way could you determine how fast the rate of mitosis is occurring in root tips? Part 3: Loss of Cell Cycle Control in Cancer • Differences between normal cells and cancer cells - Case 1: HeLa cells Case 2: Philadelphia Chromosome Part 4: Modeling Meiosis • Using model chromosomes from Part 1, explain how meiosis contributes to gamete formation and genetic diversity Part 5: Meiosis and crossing over is Sordaria • • The fungus Sordaria fimicola exchanges genetic material when two mycelia meet and fuse The resulting zygotes undergoes meiosis to produce asci; each containing eight haploid spores • Cross between wild type (+; black) and tan (tn) strains • Resulting zygote produces either parental type asci, which have four black and four tan spores in a row (4:4) • Or recombinant asci which do not have this pattern • Analysis of cross plate: • Count at least 50 asci and score them as either parental or recombinant • One map unit equals one percent recombination. The percent of asci showing recombination divided by 2 equals the map units separating the spore-color gene from the centromere. The percent of asci showing recombination is divided by 2 because only half the spores in each ascus are the result of a crossing over event. • Analysis: - Illustrate what happened during meiosis to produce the results you found How can you account for any disparities between your data and published data? Do you think the cell cycle described for mitosis could be applied to meiosis as well? Explain. Lab 8: Biotechnology: Bacterial Transformation • Question: How can we use genetic engineering techniques to manipulate heritable information? • Learning Goals: - Demonstrate the universality of DNA and its expression - Explore the concept of phenotype expression in organisms - Explore how genetic information can be transferred from one organism to another - Investigate how horizontal gene transfer is a mechanism by which genetic variation is increased in organisms - Explore the relationship between environmental factors and gene expression - Investigate the connection between the regulation of gene expression and observed differences between individuals in a population of organisms Procedure: • • • Part 1: Transformation Part 2: Analysis Part 3: Transformation Efficiency Part I: Transformation - Using a predefined procedure to transform E.coli bacteria with a plasmid carrying a foreign gene o Ampicillin resistance - Practice sterile technique • Transformed E.coli was plated on (+)amp agar and (-)amp agar. • Each plate condition was occupied by (+)E.coli and (-)E.coli. Part 2: Analysis - On which plates would you expect to find bacteria most like the original non transoformed E.coli you initially observed? - If there are any genetically transformed baterial cells, n which plate(s) would they most likely be located? - Which plates should be compared to deermine if any genetic transformation has occurred? Why? - What barriers might hinder the acquisition of plasmids? Part 3: Calculating Transformation Efficiency - Give you an indication of how effective you were in getting plasmids carrying new information into host bacterial cells. Lab 9: Biotechnology: Restriction Enzyme Analysis of DNA • Question How can we use genetic information to identify and profile individuals? • Learning Goals - Interpret restriction enzyme digest analysis - Use interpretation to solve a fictional crime Procedure • • • Part 1: Restriction Enzymes Part 2: Casting Gel Part 3: Electrophoresis Part 1: Restriction Enzymes - Naturally occurring “molecular scissors” - Typically attack foreign DNA - Specific to recognition sequence - Example: EcoRI o 5’-GA/ATTC-3’ Part 2: Casting Gel Part 3: Electrophoresis Part 4: Analysis - Calculating RFLPs - In general, DNA fragments migrate at rates inversely proportional to the log10 of their molecular weights - For simplicity’s sake, base pair length (bp) is substituted for molecular weight when determining the size of DNA fragments Part 4: Analysis - We can calculate base pair length by determining the distance of migration on a gel - A known marker is loaded (an enzyme digest that produces known bp) and can be used as a comparison to determine relative bp length of your unknown DNA samples Lab 10: Energy Dynamics • Question What factors govern energy capture, allocation, storage and transfer between producers and consumers in a terrestrial system? • Learning Goals - Design and conduct an experiment to investigate a question about how energy capture and flow in an ecosystem - Explain community/ecosystem energy dynamics, including energy flow, NPP and primary and secondary consumers - Predict interspecific ecological interactions and their effects - Use mathematical analyses in energy accounting and community modeling - Make the explicit connection between biological content and the investigative experience Procedure: • • • Part 1: Estimating Net Primary Productivity (NPP) of Fast Plants Part 2: Estimating Energy Flow Between Fast Plants Producers and Cabbage Butterfly Larvae Part 3: Independent Design Part 1: Estimating NPP - NPP is a rate energy/time - When light energy is converted to chemical energy, some energy is lost as heat during each transfer - In terrestrial ecosystems, productivity (energy capture) is measures by change in biomass of plants produced over a specific period of time o Much of an organism is water, which does not account for biomass • Model organism: Fast Plants • Design an investigation to sample biomass of an adequate number of plants early in the life cycle and then again later in the life cycle - • Plant material only…NOT WATER. Design energy transfer diagram • Analyze biomass/energy of plants early in their life cycle vs. plants late in their life cycle. • Determine average grams of biomass added per plant over the period of growth • Each gram of biomass represents about 4.35 kcal energy • Convert grams of biomass/day to NPP (kcal)/day • Reconstruct energy flow diagram Part 2: Estimating Energy Flow Between Fast Plants Producers and Cabbage Butterfly Larvae - Eat plants from the cabbage family - Energy flow into and out of these butterflies can be inferred from biomass gained and lost - Develop a diagram to model energy flow from fast plants to cabbage butterfly larvae Predict input/output of energy • Use butterflies that are into their 4th or 5th instar • First grew on yong fast plants then were transferred to Brussels sprouts - Both are part of the cabbage family • Design a procedure where you can quantify the growth of butterfly larvae over three days. • Start with freshly massed sprouts in Brassica Barn • Goal is to measure the biomass consumed by the larva, the biomass gained by the larva and the biomass lost by the larvae • Don’t forget about the energy in the frass (waste) Part 3: Independent Research Lab 11: Transpiration • Question: What factors, including environmental variables, affect the rate of transpiration in plants? • Learning Goals - Investigate the relationship among surface area, number of stomata and the rate of transpiration - Design and conduct an experiment to explore other factors, including different environmental variables on the rate of transpiration - Investigate the relationship between the structure of vascular tissues (xylem and phloem) and their functions in transporting water and nutrients in plants Procedure: • Part 1: Calculate Leaf surface area • Part 2: Design Independent Research: Transpiration Rate Part 1: Stomatal Density - How can you calculate stomatal density? Part 2: Transpiration Rate • • 2 suggested methods - Potometer - Whole plant method Potometer • Whole Plant Method Lab 12: Fruit Fly Behavior • Question What environmental factors trigger a fruit fly response? Procedure: • • Part 1: Choice Chamber Part 2: Independent Research Part 1: Choice Chamber - • • Assemble choice chamber out of tape and two water bottle Tape 20-30 flies in each chamber Allow the choice chamber to settle for 5 minutes Test a variety of chemicals/substances - Blended fruit - Alcohol - Alka-Selzter - Etc. Part 2: Independent Research - Different fruit fly mutants Chemotaxis (chemical stimulus) Phototactix (light stimulus) - Geotaxis (gravity responses) Lab 13: Enzyme Activity • Question How do abiotic and biotic factors influence the rates of enzymatic reactions? • Learning Goals: - Understand the relationship between enzyme structure and function - Make some generalizations about enzymes by studying just one enzyme in particular - Determine which factors can change the rate of an enzyme reaction - Determine which factors that affect enzyme activity could be biologically important Procedure: • • • Part 1: Developing a Method for Measuring Peroxidase in Plant Material and Determining a Baseline Part 2: Determining the Effect of pH on Enzymatic Activity Part 3: Independent Research Part 1: Developing a Method for Measuring Peroxidase in Plant Material and Determining a Baseline - • Peroxide (such as H2O2) is a toxic byproduct of aerobic metabolism Peroxidase is an enzyme that breaks down these peroxides Used by most cells in their peroxisomes Oxygen indicator - Guaiacol is a chemical that binds to oxygen in a solution and forms teatraguaiacol, which is dark brown - The more oxygen present, the darker the solution will be • Dilutions can be made to create a color palette to which we can compare relative amounts of oxygen • Color change can also be measured using a spectrophotometer • Part 2: Determining the Effect of pH on Enzymatic Activity - Use a different pH buffer for each substrate/enzyme set - Begin timing reactions of color change