Course title
BiotechnologyPre-requisite
Biotechnology ICourse description
Biotechnology II
Course Overview
This course is an extension of Biotechnology I and the career possibilities in the biotechnology field. Students will be introduced to past; present and future applications of Biotechnology and learn advanced lab protocol. Topics covered will include understanding and performance of laboratory procedures fundamental to biotechnology research which include recombinant DNA; protein purification; cell and tissue culture. An emphasis will be placed on proteins as well as independent research. Additional topics include: communication skills; the history and development of the field of biomedical research; genetically modified organisms (GMOs); bioethics; quality control; quality assurance; and career possibilities in Biotechnology.
Emphasis is placed upon laboratory skills development and data analysis. CTE standards for Biotechnology II as well as Arizona Science Standards will be followed. Students will achieve an understanding of the major principles of biotechnology through their use of problem solving skills and laboratory experiences. Students will become proficient at designing experiments; collecting data; data analysis; and problem solving skills.
Course Expectations
A foundation of knowledge from Biotechnology I is required so that students can build on their basic skills of biotechnology in the workplace and apply them to further studying advanced biotechnology techniques. Students will be expected to develop an independent research topic and conduct experiments in that area. .
Attendance:
Prompt attendance is critical for this class; as our time frame will require work from bell to bell. If you are tardy or absent you will miss critical information. Attendance and punctuality are important employability skills.
Safety
Food and drink are NOT permitted in the lab. Backpacks as well as All food and drink (including bottled water) must be left at the lab door. Proper dress for the lab includes: closed toed shoes; lab coats and goggles and gloves when necessary. All individuals have a right to an educational environment free from bias; prejudice and bigotry. ANY unsafe or disrespectful behavior will result in the immediate removal from all lab settings.
Laboratory Activities
Laboratory skills are an essential component to the course and will constitute the majority of the course. It is imperative that purpose; materials; and proper procedure are known before entering the laboratory. There will be pre- and post-laboratory discussions and student generated write-ups to help solidify the concepts. Students will learn how to use biotech equipment properly and use new techniques as they design and conduct laboratory experiments. Students will research background information and new techniques necessary to conduct a lab before experimentation. Students will complete Biotechnology II with an independent research project of their choice. Students will present their research through a research poster.
Scientific Lab Notebook
Students will maintain a legal scientific lab book that follows common biotech standard industry practices SIP. Documentation of student lab protocols; collected data and analysis will follow Good Laboratory Practice GLP.
Student Evaluation
A traditional grading scale (100-90 % = A; 89-80 % = B; 79-70 % = C; etc.) will be used for this course.
Reading quizzes and lab quizzes as well as skills tests will also be given throughout the course. Each semester grade is determined by the following:
Homework
(e.g.; completed study guides; activity worksheets;
problem sets; writing abstracts; article reviews)
Laboratory Work
(Scientific Lab Notebook)
Exams and quizzes
Final Research Project with Poster Presentation
Textbook:
Daugherty; Ellyn.. Biotechnology;. Paradigm Publishing Inc. 2007.
Student Lab Book:
Daugherty; Ellyn.. Biotechnology;. Paradigm Publishing Inc. 2007.
Proposed Course Outline Subject to Change Labs/Activities
I. Past; Present & Future Applications of Biotechnology
A. Biotechnology in the Future Potential Products Historical application and future discoveries
Scientific fields relating to biotechnology
Exploring Careers Biotech Online Search: Finding “Hot Jobs”
Bioethics: Cloning; Using Animals In Science Web Search: Monsanto & feeding the world
and Industry; Farmers & Rights Web Search: Examining a Code of Business Conduct
Activity: Design Logo for a company & write a company
Code of Business Conduct & Ethics
B. Scientific Method & Lab Safety
Lab Safety review Lab Safety Activities; Biohazard Simulations
Setting up a Scientific Notebook review Biotech Live- Biohazard (web search)
Maintaining an Inventory Inventory all glassware & maintain conditions throughout
the year.
Problem solving using the Scientific Method Lab-How Molecular Structure is Affected by Environmental
Graphing (Advanced Excel Graphing) Change (Daugherty pg 29)
Lab- SOPs Micropipette & Balance Calibration & % Error
(Protein is Cash Lab Book)
Lab-Testing a Protocol
Graphing with Excel analyzing data
Scientific Methodology in a Research Facility
Government Regulatory Agencies
C. Basic Separation & Amplification Methods
Biotechnology Equipment review Lab Using a Spectrophotometer to Check Copper Sulfate
Solutions (Daugherty Ch 3)
Introduction to Filtration
Centrifugation for Cell Study Lab- DNA extraction with a Micro-Centrifuging;
(Daugherty Ch 3)
Introduction to Bioseparations
PCR Lab-PV92
D. Laboratory Solutions
Review Concentrations Expressions & Calculations
Lab-Making Solutions of Differing Mass/Volume
(Daugherty pg 45)
Lab-Making solutions of Differing Molarity Concentrations
with the Spectrophotometer
(Daugherty pg 55)
Solutions to Support the Activity of
Biological Macromolecules Lab-Making Media for Bacteria Cell Culture (Media Prep)
Document the Preparation & Storage of all Solutions (Daugherty pg 71)
Culture Media for Competent Cells Lab-Bacterial Cell Culture
(Daugherty pg 76)
Lab-Growing & Maintaining Healthy Cultures: Effects of
Chemicals & UV light on Bacterial Cultures
E. Quality Assays and Tests
The Components of an Assay Lab- Searching for Native Amylase Producing Bacteria
(Daugherty Ch 6)
Model Organisms Lab: Bioreactor (Simonson; Grimes; King)
Assay Development
Quality Tests & Assays Lab: Making Bread Using Yeast as a Model Organism
Quality Assurance (Pima County JTED)
Quality Control Lab: Quality Assurance in Manufacturing a Product (Austin Community College)
Technology Relating to Assays & Examples Lab-Testing Plant & Animal Samples for Hydrogen Peroxidase
(Daugherty pg 120 Ch 6)
Lab-Got Protein? (Bio-Rad)
Using the Spectrophotometer Lab- Using the Spectrophotometer to Measure Protein
Constructing a Standard Curve Concentration & Enzyme Activity
(BioRad Biofuels)
F. Biotech Skill Standards Maintain Standards Mastery through Standards Skills Booklet
II. Introduction to Studying Proteins
A. Protein Structure 5
Protein Sequence & Mutations
Classes of Proteins: Structure & Function Lab-Chromatography-Separation of Proteins (BioRad) Proteins & Transport Across the Membrane Lab: Western Blot (BioRad) Proteins used in Biotechnology Plasmid Technology Lab-Transformation DNALC Green Gene Colony
Transformation Kit Refill
(Carolina Refill 4-8 station # RC-211082C 21.00)
B. Enzymes 5; 7
Reaction Rates Lab-The Action of Different Enzymes on Apple Juice
Concentration Production (Daugherty Ch 5)
Substrates Lab: Biofuels (BioRad)
Effects of Temperature
C. Advanced Protein Studies: Protein PAGE & Purification
Native & SDS Page Lab – Comparative Proteomics (BioRad)
Phylogeny Lab- Characterizing Proteins by PAGE
Vertical Gels (Daugherty Ch 5) Lab-Separating and Identifying Proteins via SDS PAGE
(Daugherty Ch 5)
pGLO Kit SDS-PAGE Extension w/ vertical gel
(BioRad 166-0013EDU 85.00)
Separating Proteins using Ion Exchange Lab-GFP Chromatography Kit
Chromatography (BioRad: # 166-0005EDU 101.00)
D. Studying Antibodies 5
Antibodies Lab-Protein Separation and Identification
Structure & Function Lab-ELISA (BioRad)
Immunology
Vaccines
Biomanufacturing Proteins Thinking Like a Biotechnician
E. BIOINFORMATIC RESOURCES Identify & Utilize electronic databases/websites (NCBI)
To search for relationships between protein
Sequences http://www.ncbi.nlm.nih.gov/
III. Biotechnology in Medicine
A. Identifying a Potential Biotechnology Product 6. 8; 9
Sources of Potential Products Biotech Online-Amazon Hide and Seek
Bringing a Biotechnology Product to Market Activity: R & D for new Product: Calculate Cost;
Product Quality Control Target Market; Time Looking for New Products in Nature Lab-Isolating Horseradish Peroxidase from
Radish Root (Daugherty Ch 6)
B. Drug Discovery 12
Using a Spectrophotometer to Detect Molecules Lab-Better Use of the Spectrophotometer
Using the Spectrophotometer to Measure Protein Lab-Determining the Purity & Concentration of DNA
Concentration Samples (Daugherty Ch 11)
Using the Spectrophotometer in Medicine
C. Recombinant Biotechnology 8
Creating Pharmaceuticals by ProteiN/Antibody Lab-Immunodetection Procedures to Detect Myosin
Engineering Light Chain(BioRad )
Overview of Genetic Engineering Lab: Rainbow Plasmids
Plasmid Technology
D. Plant Biotechnology 10; 11
Basic Plant Propagation Lab-Asexual Plant Propagation through Leaf & Stem
Cuttings (Daugherty Ch 11)
Lab-Wisconsin Fast Plants: How fast is fast?
(Daugherty Ch 10)
Basic Plant Structure; Growth & Development
Cloning by Plant Tissue Culture Lab-GMO Investigator Kit
GMO are they a good thing? (BioRad )
Plant Tissue Culture Activity: Patents on GMOs (web search)
Applications of Biotechnology in Agriculture & Horticulture
Isolating DNA from Plant Cells Lab-DNA extraction from Plant Cells with Gel Electrophoresis
E. Bioethics
Cloning
Stem Cells http://learn.genetics.utah.edu.
Eugenics Web based exercise on Cloning
Gene Therapy Web-Eugenic Movement
Legal vs Illegal/Ethical vs Unethical Pima county JTED
Human Genome Project DNALC
E. BIOINFORMATIC RESOURCES Identify & Utilize electronic databases/websites (NCBI)
BLAST To search for relationships between protein
GenBank Sequences http://www.ncbi.nlm.nih.gov/
PubMed Lab: What is NCBI (Dr. David Andrew UofA)
OMIM Lab: Nucleotide Sequence Alignment
(Dr. David Andrew UofA)
Lab: Human Diseases Mystery Diseases
(Dr. David Andrew UofA)
V. Advanced Biotechnology Independent Research
To be started 2nd semester; possible topics 14
DNA Sequencing
Genomics
Nanotechnology
Cloning
Plant/Animal Cell Cultures
DNA Barcoding
Agrobacterium
C. elegans
Final Project-Research Poster & Presentation
Dress for Success
Sample Lab
Does molecular evidence support the theory of evolution?
The Human Genome Project has completed the task of sequencing all human genes. Far from closing the book; this body of work opened up a whole new field.
Proteomics asks the question: What do our genes do? Genes encode proteins that determine an organism’s form; function and phenotype–the raw material of natural selection. Proteomics is the study of the structure; function; and interaction of proteins with each other and with their environment.
The Protein Profiler moves beyond DNA and allows students to employ protein electrophoresis; the most widely used technique in life science research; to study protein structure and function. Students learn to use SDS-PAGE to generate protein profiles from the muscles of both distantly and closely related species of fish. From their results they compare the different species profiles to test the hypothesis that protein profiles can be indicators of evolutionary relatedness.
This kit allows your students to explore evolution at the molecular level within the context of the central molecular framework of biology:
DNA > RNA > Protein > Trait = Phenotype
Changes in proteins can reflect changes in the gene pool. Muscle consists mainly of actin and myosin; but muscle tissue comprises numerous other proteins. Actin and myosin are the major muscle proteins essential for locomotion and survival in all animals. As such; the structures and functions of actin and myosin have remained relatively stable “or conserved” in all animals over evolutionary time. However; other muscle proteins exhibit considerable variation even among closely related species. Detectable variations between organisms’ protein profiles reflect physiological adaptations to different environments; but they originate as random DNA mutations. Such mutation events; if favorable; persist through the natural selection process and contribute to the evolution of species–with new specialized functions.
Mutation > Variation > Specialization > Speciation = Evolution
This is an open-ended inquiry-based kit. Students make predictions about their results in pre-lab activities using internet databases and published phylogenetic information. They generate novel results and apply their findings directly to the problem of solving evolutionary relationships by constructing cladograms (phylogenetic trees). From their gel data; they build up a tree and assign each organism a branch. Students can decide whether their results support their predictions.
The kit guides students through the thought processes involved in a laboratory-based scientific investigation. Students are asked: Can molecular evidence support the theory of evolution? Why or why not? What explanations can you suggest?
Lesson 1: Protein Extraction From Muscle
Your first task is to extract proteins from muscle tissue; unfold and denature them; and give each protein an overall negative charge using Laemmli sample buffer; mechanical forces; and heat. In this lab you will add tiny pieces of muscle to Laemmli sample buffer and manually disrupt the tissue by flicking the tubes. This will release muscle specific proteins from the cells; unfold them; and add an overall negative charge to each protein. You will then pour off the extract and heat the extracted proteins to 95°C; which will complete their denaturation.
Student Workstations
Material Quantity
1.5 ml fliptop microtubes 5
1.5 ml screwcap microtubes 5
1 ml transfer pipet 1
Fish samples; labeled 1–5 5 species
Marking pen 1
Laemmli sample buffer 1.5 ml
Knife or scissors to cut fish samples 1
Common Workstation
Material Quantity
Water bath set to 95°C 1
Procedure
1. To make this a blind study; assign a letter (e.g.; A–E) to each fish sample to be investigated. Keep a record of which fish got which number and hide their true identities until after the analysis is complete.
2. Label 1.5 ml fliptop microtubes with the number of the fish species to be analyzed.
There should be one labeled tube for each fish sample being prepared for electrophoresis.
3. Add 250 µl of Laemmli sample buffer to each labeled tube.
4. For each sample; obtain a piece of fish muscle (avoid skin; fat; and bones) approximately
0.25 x 0.25 x 0.25 cm3 ( ); and transfer it to the appropriately labeled microtube. Close the lid.
Student Manual
5. Gently flick the microtube 15 times with your finger to agitate the tissue in the sample buffer.
6. Incubate the samples for 5 min at room temperature to extract and solubilize the proteins.
7. Pour the buffer containing the extracted proteins; but not the solid fish piece; to a labeled 1.5 ml screwcap tube. Note: It’s not necessary to transfer all of the fluid to the screwcap tube; since only a small volume (
8. Heat your fish samples in their screwcap tubes for 5 min at 95°C to denature the proteins in preparation for electrophoresis.
9. Store the samples at room temperature if they are to be loaded onto gels within 3–4 hr; or store them at –20°C for up to several weeks.
Student Manual
Lesson 1 Focus Questions
1. Why did you add Laemmli sample buffer to your fish samples?
2. What was the purpose of heating the samples?
3. How are the proteins extracted from the fish samples?
4. Have all the proteins been extracted from the fish slice or are some still left after the extraction? How could you test your hypothesis?
Student Manual
Lesson 2: Electrophoresis: Gel Loading; Running; and Staining
So far you have extracted; denatured; and given the proteins from fish muscle tissue a negative charge. Now they can be separated according to their molecular weights using gel electrophoresis; which will generate profiles for various fish species
Student Workstations
Material Quantity
Fish protein extracts from lesson one 5 species
Actin & myosin standard; 12.5 ul 1 vial
Precision Plus Protein Kaleidoscope prestained standards; 6 µl 1 vial 4-20% Mini-PROTEAN TGX or 15% 10-well; Ready Gel precast gel 1 vial 1–20 µl adjustable-volume micropipet 1
Prot/Elec pipet tips for gel loading 7 tips
Mini-PROTEAN Tetra cell electrophoresis module 1 per 2 gels
1x Tris-glycine-SDS (TGS) running buffer 700 ml per gel box Power supply (200 V constant) to be shared between workstations 1
Sample loading guide – for 10-well comb (optional) 1 per gel box
Buffer dam (only required if running 1 gel/box) 1
Staining trays 1 per 2 gels
Bio-Safe Coomassie stain for proteins 50 ml per 2 gels
Common Workstation
Material Quantity
Water bath set at 95°C 1
Water for gel destaining (tap water is fine)
Student Manual
Procedure
1. Reheat frozen fish samples and actin and myosin standard at 95°C for 2–5 minutes to redissolve any precipitated detergent. Note: If you have prepared your fish samples in this lesson; there is no need to reheat them.
2. Assemble gel boxes. Use the pictorial guide found in the Quick Guide to insert your TGX or Ready Gel polyacrylamide gels into the vertical electrophoresis module if your instructor has not preassembled them.
3. Double-check that the buffer in the inner buffer chamber is well above the top of the smaller plate. If it is not; you may have a leak; consult with your instructor.
Note: If you do have a leak; the outer chamber of the gel box can be filled to above the inner small plates; to equalize the buffer levels in both reservoirs.
4. If available; place a yellow sample loading guide on the top of the electrode assembly.
The guide will direct your pipet tip to the correct position for loading each sample in a well.
5. Record in which well of your gel you will load which of your samples in the table below:
Well
1 Volume
empty Sample Name
none
2 empty none
3 5 µl Stds Precision Plus Protein Kaleidoscope prestained standard (Stds)
4 *10 µl sample A
5 *10 µl sample B
6 *10 µl sample C
7 *10 µl sample D
8 *10 µl sample E
9 *10 µl AM actin & myosin standard (AM)
10 empty none
*Note: If you are going on to perform the western blot module load 5 µl of each fish sample and the actin & myosin standard. This will prevent overloading the lanes.
Student Manual
6. Load 5 µl of Precision Plus Protein Kaleidoscope prestained standard gently into well
# 3 using a thin gel loading tip. Note: The fine barrel of the gel loading tips means liquid is slower to go into the tip than normal tips. You must therefore release the plunger of the micropipet very slowly; otherwise you will not pipet the correct volume.
7. Using a fresh tip each time; load 10 µl of each of your protein samples gently into the wells designated in your table above. Note: If you are going on to perform the western blot module; load 5 µl of each protein sample.
8. Using a fresh tip; load 10 µl of the actin & myosin standard gently into well # 9. Note: If you are going to perform the western blot module load 5 µl of the actin & myosin standard.
9. After loading all samples; remove the yellow sample loading guide (if used); place the lid on the tank; and insert the leads into the power supply; matching red to red and black to black. Set the voltage to 200 V and run the gels for 30 minutes. Watch for the separation of the standard.
10. When gels are finished running; turn off the power supply and disconnect the leads.
Remove the lid and lift out the electrode assembly and clamping frame.
11. Pour out the running buffer from the electrode assembly. Open the cams and remove the gel cassettes.
12. Now it’s time to stain the proteins in your gel. Lay your gel cassette flat on the bench with the short plate facing up. Carefully pry apart the gel plates; using the gel opening key. The gel will usually adhere to one of the plates. Transfer the plate with the gel adhering to it to a tray containing tap water allowing the liquid to detach the gel from the plate. The gel may also be lifted directly (and gently!) from the plate and placed into the water. If there is sufficient time; rinse the gel 3 times with tap water for 5 minutes by carefully pouring out the water and replacing it. Rinsing the gel will improve the intensity of the protein bands.
13. Carefully pour out the water and replace with 50 ml of Bio-Safe Coomassie stain per 2 gels.
Student Manual
14. Allow the gels to stain for at least 1 hour; with shaking if available. Gels maybe stained overnight but seal the container to reduce evaporation.
15. After at least 1 hour discard the stain and replace it with a large volume of water to destain the gel overnight with rocking action if available. Change water 2–3 times if possible. Bands will become visible after a few hours of destaining.
Lesson 2 Focus Questions
1. Why do SDS-coated proteins move when placed in an electric field?
2. What is the purpose of the actin & myosin standards and the Precision Plus Protein Kaleidoscope prestained standard?
3. Which proteins will migrate farthest? Why?
4. What is the purpose of the stain?
School country
United StatesSchool state
ArizonaSchool city
ScottsdaleSchool / district Address
8500 E. Jackrabbit RoadSchool zip code
85018Requested competency code
Lab ScienceDate submitted
Approved
YesApproved competency code
- LADV
- Advanced science
- LINT
- Integrated science