|ETP Title:||Nitrogen Cycling by Soil Microbes|
|Organization:||Francis Lab, Stanford University|
This lesson series will introduce students to nitrogen cycling by soil microbes. The content area will cover microbiology, DNA analysis, and the nitrogen cycle. Lab techniques include serial dilutions, bacterial plating and sterile technique, micropipetting, PCR and gel electrophoresis. Students will collect soil samples and plate them on nitrogen-free (selecting for nitrogen-fixing bacteria) and nutrient agar using direct soil and/or serial dilution techniques. Students will examine the plates visually and use a bacteria/fungi dichotomous key to draw hypotheses concerning identification.From these results, students will isolate bacterial colonies and streak plates to clone the bacteria. Students will run PCR using primers for bacterial amoA, ammonia monooxygenase, an enzyme responsible for converting ammonium to nitrite and nitrate (found in nitrifying bacteria), and 16S rRNA (present in all bacteria). PCR products will be analyzed by gel electrophoresis. Students will compare their initial identifications with results from the PCR to determine if nitrogen-fixing bacteria are also responsible for ammonia oxidation, and will position the bacteria they find in the nitrogen cycle. Lessons 1 and 2 together can stand alone, in the event that teachers don't have access to PCR and gel electrophoresis equipment. Lesson 3 is an extension using PCR and gel electrophoresis techniques.
California StandardsCell Biology 1. The fundamental life processes of plants and animals depend on a variety of chemical reactions that occur in specialized areas of the organism's cells. As a basis for understanding this concept: b. Students know enzymes are proteins that catalyze biochemical reactions without altering the reaction equilibrium and the activities of enzymes depend on the temperature, ionic conditions, and the pH of the surroundings. Genetics 4. Genes are a set of instructions encoded in the DNA sequence of each organism that specify the sequence of amino acids in proteins characteristic of that organism.
Ecology 6. Stability in an ecosystem is a balance between competing effects. As a basis for understanding this concept: d. Students know how water, carbon, and nitrogen cycle between abiotic resources and organic matter in the ecosystem and how oxygen cycles through photosynthesis and respiration.
Investigation & Experimentation - Grades 9 To 12 Science Content Standards. 1. Scientific progress is made by asking meaningful questions and conducting careful investigations. As a basis for understanding this concept and addressing the content in the other four strands, students should develop their own questions and perform investigations. Students will: a. Select and use appropriate tools and technology (such as computer-linked probes, spreadsheets, and graphing calculators) to perform tests, collect data, analyze relationships, and display data. d. Formulate explanations by using logic and evidence. j. Recognize the issues of statistical variability and the need for controlled tests. l. Analyze situations and solve problems that require combining and applying concepts from more than one area of science.
National Board StandardsEstablishing a Favorable Context for Student Learning IV. Engaging the Science Learner (p. 27) Accomplished Adolescence and Young Adulthood/Science teachers spark student interest in science and promote active and sustained learning, so all students achieve meaningful and demonstrable growth toward learning goals.
VII. Fostering Science Inquiry Accomplished Adolescence and Young Adulthood/Science teachers engage students in active exploration to develop the mental operations and habits of mind that are essential to advancing strong content knowledge and scientific literacy.
Objectives for Science Content (California Science Standards in parentheses):
- Students will know that nitrogen cycling is largely accomplished by soil microbes. (Ecology 6.d.)
- Students will know that the reactions in the nitrogen cycle are catalyzed by enzymes (proteins) in the microbes. (Cell Biology 1.b.)
- Students will know that microbes are studied and categorized mostly by analyzing DNA sequences of genes. (Genetics 4.)
Objectives for Investigation and Experimentation (California Science Standards in parentheses):
- Students will be able to write and test hypotheses using the technology available for experimentation, and draw conclusions from resulting data. (Investigation & Experimentation Grades 9 to 12 - 1.d.,and 1.j.)
- Students will be able to perform serial dilutions, bacterial plating, micropipetting, and PCR and gel electrophoresis. (Investigation & Experimentation Grades 9 to 12 - 1.a.)
- Students will be able to synthesize content knowledge from cell biology, genetics, and ecology to explain the influence of soil microbes in the nitrogen cycle. (Investigation & Experimentation Grades 9 to 12 - 1.l.)
Worksheets with questions are included for each lesson.
Quizes are included for Lessons 1 and 2
The connection between the ETP and Fellowship. :
My fellowship is in the department of Environmental Earth System Science. I will be collecting sediment samples from the San Francisco Bay with varying salinity concentrations. I will extract DNA from these samples and, using PCR, amplify genes for archael amoA, ammonia monooxygenase, an enzyme responsible for converting ammonium to nitrite and nitrate. The PCR product will then be cleaned, cloned, and sent for sequencing. Sequences returned will be checked through BLAST and the NCBI database and used to create a phylogenetic tree.This process is quite similar to the student lab I will be developing. I plan to partner with the East Bay Biotechnology Education Program http://www.ebbep.org this fall to borrow PCR equipment for the classroom.
Required Content Knowledge:
- Cell biology and biochemistry
- Genetics and DNA technology (PCR and gel electrophoresis)
- Introduction to ecology
Lessons 1 and 2 can be stand-alone lessons together if PCR and gel electrophoresis equipment is not available.
The following websites are good introductions to sterile technique, which is necessary to teach prior to engaging in this lesson series:
Lesson 1 – (3 days) Introduction to soil, soil microbiology, and collecting soil samples.
In this activity, students are introduced to soil, soil microbiology, and the nitrogen cycle. They are instructed in appropriate techniques for soil sampling. A PowerPoint presentation leads the students through all 3 days of instruction. There are student worksheets for each day (3 total) and a Quiz. Attachments for Lesson 1: 6 total.
Lesson 2 – (2 days) Day 1: Serial dilutions and plating (incubate overnight at 37 C); Day 2: Examine plates and identify with dichotomous key; Isolate bacterial colonies and streak plates to created colonies for PCR.
In this activity, students will culture bacteria on two types of medium - Nitrogen-free Ashby's glucose agar, and LB Nutrient agar. Nitrogen-fixing bacteria will likely not grow well on the LB nutrient agar, but will have an advantage on the selective nitrogen-free medium. Students will draw hypotheses about what types of bacteria will grow on each medium and will test their hypotheses using a dichotomous key. Students will select colonies and streak plates to create colonies for Lesson 3 - PCR and gel electrophoresis. There is a student worksheet attached, along with a dichotomous key and a Quiz. Attachments for Lesson 2: 4 total.
Lesson 3 – (2 days) Set-up PCR for amoA and 16S rRNA, and examine results with gel electrophoresis.
In this activity, PCR will be done on selected colonies for amoA, an enzyme responsible for ammonia oxidation in the nitrogen cycle. Students will hypothesize whether bacteria responsible for nitrogen-fixation, which they have selected for and identified in Lesson 2, are also responsible for ammonia oxidation, another step in the nitrogen cycle. PCR will also be done for 16S rRNA, which is present in all bacteria, to validate that colonies were of bacterial origin. There is a student worksheet, and 2 excel files to aid the teacher in preparing for PCR. Attachments for Lesson 3: 4 total. pre
Bibliographic or other resources you used in creating this curriculum:
Francis, C. A., K. J. Roberts, J. M. Beman, A. E. Santoro, and B. B. Oakley. 2005. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proceedings of the National Academy of Sciences 102:14683-14688.
Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: A model for molecular microbial ecology. Annual Review of Microbiology, 55: 485-529.
Mosier, A.C., and Francis, C.A. (2008) Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary. Env. Microbiology 10(11), 3002–3016.
The needs this ETP will fulfill in the classroom, teaching or school:
The extent that I have been able to incorporate microbiology into my curriculum was to plate collected swabs of various surfaces in the school or classroom on nutrient agar and observe growth. I had no technology element in my microbiology unit. I especially wanted to run PCR and gel electrophoresis, but did not have access to the equipment or appropriate curriculum. Also, there were particular lab skills that I wanted to have in my curriculum. Namely, serial dilutions and micropipetting. This series of lesson plans meets all of these goals, and has an added bonus of combining microbiology with DNA analysis, which meets content standards requirments. It also is based on one process of nitrogen cycling in the environment, which is an important part of my ecology unit. It will suit the needs of my AP biology, regular biology, and ITA biology classes (Information Technology Academy). It will also situate nicely into a year-long stream monitoring program that I will be implementing this year - one that will move my teaching towards project-based curricula.
Keywords:Microbe, Bacteria, Nitrogen Cycle, Soil