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The Nitrogen Cycle

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The Nitrogen Cycle SEQUENCE CSI: THE NITROGEN CYCLE Stephanie Wilson Virginia Institute of Marine Science Grade Level High School Subject area Biology The VA SEA project was made possible through initial funding from the National Estuarine Research Reserve System Science Collaborative, which supports collaborative research that addresses coastal management problems important to the reserves. The Science Collaborative is funded by the National Oceanic and Atmospheric Administration and managed by the University of Michigan Water Center. VA SEA is currently supported by the Chesapeake Bay National Estuarine Research Reserve, Virginia Sea Grant, and the Virginia Institute of Marine Science Marine Advisory Program. Title Sequence CSI: The Nitrogen Cycle Focus Molecular techniques: Identifying organisms within the nitrogen cycle. Topics covered: o Technology in genetic/molecular analysis o DNA, sequencing o Gel electrophoresis o Cycles / relationships Grade Levels/Subject High School Biology (9-12) VA Science Standards - BIO.1 i) appropriate technology including computers, graphing calculators, and probeware, is use for gathering and analyzing data, communicating results, modeling concepts, and simulating experimental conditions; - BIO.5 g) the structure, function, and replication of nucleic acids; - BIO.5 j) exploration of the impact of DNA technologies; - Bio.8 b) nutrient cycling with energy flow through ecosystems; Learning Objectives - Students will apply practical scientific molecular methodology in the classroom to solve a problem. - Students will utilize gel electrophoresis pictures in order to identify samples. - Students will identify DNA sequences using a practical, scientific database: NCBI. Total length of time required for the lesson 45 minutes to 1 hour Key words, vocabulary - DNA: Deoxyribonucleic acid, the double-stranded carrier of genetic information, is present in almost all living organisms. It genetically determines the makeup of an organism such as their hair or eye color. There is a unique code for every organism to make up the double helix of DNA. - DNA Sequence: The code that makes up DNA is its sequence, it consists of four nucleotides adenine (A), cytosine (C), guanine (G), and thymine (T). The order of the nucleotides codes for specific proteins and characteristic in an organism. - Gel Electrophoresis: A molecular analysis technique that separates DNA out by size using an electric charge. Depending on how long a sequence is, how many basepairs or nucleotides are in Virginia Scientists and Educators Alliance (VA SEA) 2019 Wilson Page 1 of 31 it, it will move a certain distance along an agarose gel. DNA is a negatively charged molecule and therefore it can be pulled through a gel with smaller DNA fragments moving further than larger ones. - Gene: A gene is encoded by a section of an organism’s DNA, genes will give rise to characteristics in an organism and can be heritable (passed from parent to offspring). - Nitrogen Cycle: The nitrogen cycle is the movement of nitrogen throughout an ecosystem – the full nitrogen cycle moves nitrogen through the geosphere, the atmosphere, and the biosphere. Nitrogen can change forms, for example it can be a gas, or it can be fixed to form ammonium, or it can be found in other larger compounds such as amino acids. - Scientific Standard: A standard is a known measurement that can be utilized in scientific analyses; the standard is a reference point that is used to calibrate sample measurements. - Sequencing: A molecular analysis technique in which the code of DNA, made up of its nucleotide sequence, is read. This can be used to identify organisms because each organism has a unique sequence. - Sequence Database: A database that can be used to compare sequences to those that have already been identified in order to identify an unknown. These databases can also be used to search for certain sequences. Background Information See background PowerPoint. My research focuses on biogeochemistry and, more specifically, the cycling of nitrogen in coastal ecosystems. To answer questions about the nitrogen cycle, I do a lot of molecular analyses to identify and quantify microorganisms because the majority of the nitrogen cycle is mediated by microbes. To identify microorganisms, I utilize DNA sequencing of a gene called the 16S ribosomal RNA gene that is present in all bacteria and archaea. This allows me to compare gene sequences to databases of previously identified organisms and identify the organisms in my own sample. Identifying which organisms are present can allow me to infer what potential biogeochemical reactions may be occurring in the environment. Nitrogen and its cycling is extremely important; all organisms require nitrogen, it can promote primary production in aquatic environments where it can be a limiting nutrient for phytoplankton or as fertilizer in agriculture, and it can be a pollutant when in excess for example when there are high levels of nitrogen in wastewater effluent. This lesson focuses on identification of organisms using their DNA, because every organism has a genetic code. Using biotechnology, scientists can identify organisms to help them answer questions about the environment. For example, gel electrophoresis which separates DNA fragments by size can help to identify organisms. DNA sequencing is another avenue of identification in which previously identified organisms whose sequences have been deposited in databases can be used to identify unknown samples. These techniques are used in cutting edge research labs for identifying organisms, examining genetic variation, and assessing ancestry. Once organisms are identified relationships between them can be assessed. For example, most of the nitrogen cycle is completed Virginia Scientists and Educators Alliance (VA SEA) 2019 Wilson Page 2 of 31 by microorganisms. Identifying which microorganisms are present in a sample can allow scientists to make inferences as to what cycling reactions may occur. Handouts and references: see table below -Envelopes to put “samples” in 1. CSI Sequence: Sequence Identification Worksheet (App. 1a) 2. Gel Electrophoresis Results (App. 2a-d) 3. NCBI Search Sequences (App. 3a, 4 sequences) & KEY (App.4) 4. How to Use NCBI website & Nucleotide BLAST Search – Instructions (App.4) 5. CSI Sequence: Connections Worksheet (App. 5a) 6. CSI Sequence: Organism Information Sheets (App. 6a-d) Title For Students (worksheets & references) For Teacher (references or KEY) 1. CSI Sequence: Sequence Appendix 1a: Appendix 1b: Identification Worksheet Teacher’s ANSWER KEY for Student worksheet Sequence ID worksheet 2. Gel Electrophoresis Results Appendices 2a – 2 d: Appendices 2a – 2 d: Sample A Sample A = bacteria Sample B Sample B = bacteria Sample C Sample C = plant Sample D Sample D = mammal 3. NCBI Search Sequences Appendices 3a (4 sequences): Appendices 4 (4 sequences): Better to have these already loaded on computers or on a flash drive so Sample A Teacher’s ANSWER KEY for that students can copy and paste Appendices 2a – 2 d Sample B their assigned sequence into the Sample A = N-fixing bacteria NCBI website Sample C Sample B = Denitrifying bacteria Sample D Sample C = Soybean plant Sample D = Cow, mammal 4. How to Use NCBI website & Appendix 3b the Nucleotide BLAST Search 5. Organism Information Sheets Appendices 5 a – d: Sample A = Rhizobium lemnae (N-fixing bacteria) Sample B = Rhodanobacter denitrificans (denitrifying bacteria) Sample C = Glycine max (soybean) Sample D = Bos taurus (cow) 6. Sequence Connections Worksheet Appendix 6a: Appendix 6b: Teacher’s ANSWER KEY for Student worksheet Sequence Connections worksheet Virginia Scientists and Educators Alliance (VA SEA) 2019 Wilson Page 3 of 31 Materials and supplies -Projector/Screen for introductory PowerPoint -Computer for each team (or each student) to access NCBI Website -NCBI Website: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=bla sthome Classroom Set-Up Tables arranged in groups for teams of 2-4 students. Procedure Introduction: Attention Grabber: - Have any of you ever written in code? Or have you ever had to decipher a code to see what it means? - The PPT has a code they can try to decode - The introduction PowerPoint can be used to review concepts about DNA. - This will also be an introduction to molecular techniques such as gel electrophoresis and DNA sequencing. - PowerPoint Script: Slide 1: Presentation Title Slide 2: Attention Grabber Students should try to decode the message – once they figure it out you can relate this to how DNA encodes information, just like the code they read! Slide 3: My research These are picture of me in the lab and in the field: I am a graduate student that studies the nitrogen cycle in coastal environments. In these pictures I am exploring the nitrogen cycle in the lab and in the field. The first picture is a picture of me at my study site with some of my colleagues we are collecting water and sediment samples in order to look at biogeochemical processes occurring at this beach; The Gloucester Point Beach in Virginia. The second photo is one of me in the lab performing molecular analyses on my samples from the field; here I am extracting DNA! This DNA will be used to determine what organisms are living in the water and sand of the Gloucester Point Beach. Slide 4: DNA is like a code! Almost every organism on the planet has DNA. It encodes the genetic material of the organism and serves as the blueprint for the organism’s characteristics. Each organism’s genetic code is unique and therefore they can be identified using that code – for Virginia Scientists and Educators Alliance (VA SEA) 2019 Wilson Page 4 of 31 example DNA from crime scenes can be used to identify suspects or DNA from an organism can be used to identify what kind of organism it is. Slide 5: DNA Review Remember that DNA stands for deoxyribonucleic acid and it is a double stranded molecule. The code is made up of four nucleotide bases Adenine, Thymine, Cytosine, and Guanine. Does anyone remember which bases pair to with each other? A-T and C-G! The two strands of DNA, made up of these bases, are held together by hydrogen bonds.
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