Course title
BiotechnologyPre-requisite
C or better in Biology and Algebra I and/or teacher recommendation. Chemistry or Concurrent enrollment in Chemistry recommendedCourse description
Course Overview
This course is an introduction of current biotechnology and applications in everyday life as well as the career possibilities in the biotechnology field. Students will be introduced to past; present and future applications of Biotechnology and learn proper lab procedures. Topics covered will include understanding laboratory procedures fundamental to biomedical research which include recombinant DNA; protein purification; cell and tissue culture. Additional topics include communication skills; the history and development of the field of biomedical research and understanding the legal environment and technology transfer aspects of biomedical research.
Emphasis is placed upon laboratory skills and data analysis. Students will achieve an understanding of the major principles of biotechnology through their use of problem solving skills. Students will become proficient at collecting and analyzing data using professional laboratory equipment.
Course Expectations
A foundation of knowledge from introductory biology and basic biochemistry is assumed so that most of the class time can be spent learning; reinforcing and applying biotechnology concepts in a laboratory setting. Students are expected to complete reading assignments from the textbook prior to class. Students must also keep a legal scientific lab book of all experiments.
Attendance:
Prompt attendance is critical for this class; as our time frame will require work from bell to bell and you will miss critical information if you are not present and ready to work when the bell rings. Prompt and regular attendance are critical employability skills practiced in this course.
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
Scientific Lab Notebook
Students will maintain a scientific notebook 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.
A test will be given for each major concept. The test will be modeled after the CTE Bioscience standards and the Arizona state science standards. Students will demonstrate their knowledge of the concepts through written exams as well as performance of lab skills. Throughout the course reading and lab quizzes may be given as well as laboratory skills tests. Each semester grade is determined by the following:
Textbook:
Daugherty; Ellyn.. Biotechnology;. Paradigm Publishing Inc. 2007. ISBN 0-7638-2282-5
Student Lab Books:
Daugherty; Ellyn.. Biotechnology;. Paradigm Publishing Inc. 2007. ISBN 978-76582-902-5
Simonson; Nina Xan; Grimes; Amanda; Remenih; Christine.. A Dynamic Natural Approach to Instruction
Biotechnology.; Kendall Hunt Publishing 2011. ISBN 978-0-7575-9365-9
Proposed Course Outline Subject to Change Labs/Activities Chapters
I. Past; Present & Future Applications of Biotechnology (Daugherty)
A. Historical Applications of Biotechnology Timeline-Evolution of Biotechnology 1
Variety of Products Replicate historical application of
Scientific fields relating to biotechnology biotechnology (e.g.; cheese; yogurt)
Exploring Careers Lab: Making Cheese
Bioethics Bioethics-Stop You Can’t Use Those Cells!
Field trips; guest speakers; web searches
B. Standard Lab Operating Procedures (SOP) Scientific Method & Lab Safety 1
Lab Safety: Safety Symbols & Practices Lab Safety Activities
Emergency Protocols
Setting up a Scientific Notebook Biotech Live- Biohazard (web search)
Problem solving using the Scientific Method Lab: Using the Scientific Method -Blue Jean lab
Graphing (Intro to Excel Graphing) Lab-Scientific Methodology in a Research Facility- Biotech Acronyms Graphing with Excel & Creating a Logo & poster)
Standard Operating Procedures SOPs SOPs: Cleaning a Pipette; % error on balance & Pipette
Good Laboratory Practices GLPs
Biotech Skill Standards
Micropipetting Lab: Pipette Practice
Lab: Pipette Challenge
II. Biochemistry
A. Cells-Characteristics of Model Organisms Used in Biotechnology 2
Prokaryotes vs Eukaryotes
Structure & Function of Organelles Lab-Cell Study-intro to centrifuge
Mitochondria Lab-Diffusion Across the Membrane
Transport Across the Membrane Lab-Better Microscope Technique
Levels of Organization
Cells Used in Biotechnology Web search: CHO; Ecoli; HeLa; Yeast
Central Dogma of Biology Working Safely with Bacteria
Macromolecules
Carbohydrates
Lipids Lab-Identifying Carbohydrates; Lipid & Proteins
Proteins
Nucleic Acids DNA Extraction Exercise
Plasmids
Cellular Energetics
B. Basic Skills of the Biotechnology Workplace 3
Using a balance
Measurement with Metrics & Conversions Measurement Lab
Measuring Mass & Volume
Compounds Lab-Learning to Use the Spectrophotometer
Solutions Lab-Making Solutions
pH & Buffers mass/volume; % mass/volume; Molarity
Hydrophobic/Hydrophilic Lab-Using a Spectrophotometer to identify concentrations
of Methylene Blue [MB]
Molarity Lab-Serial Dilutions
Dilutions
Aseptic Procedures in Lab Lab-Making & pouring Agar plates
Laminar flow hood plating & swabbing lab station
UV lights; Bleach & 70% IPA
C. Studying DNA-Structure; Function; Isolation & Analysis 4
DNA Structure & Function Lab-Making a DNA model
Lab: Human Check Cell DNA Extraction (1 day)
Sources of DNA (BioRad DNA in a Bottle)
Prokaryotes and Plasmids Lab-Bacterial Cultures
Eukaryotic DNA Lab-Gram Staining Bacteria
Viral DNA Lab: DNA Goes to the Races & Restriction
Enzymes (1.5 days)(Carolina)
Isolating & Manipulating DNA Lab-DNA Extraction from Bacteria
Using Gel Electrophoresis to Study DNA Lab-Gel Electrophoresis
D. Studying Proteins-Structure; Function; Isolation & Analysis 5
Structure and Function of Proteins Lab-Making a Protein
Protein Synthesis Lab-Protein Separation and Identification
Using Biuret solution
Mutations Lab-Mutations
Protein Catalysis Lab-Enzymes
Applications of Protein Analysis
Biomanufacturing Proteins Thinking Like a Biotechnician
Bioethics-Who Owns the Patent on the Genetic Code?
E. BIOINFORMATIC Identify & Utilize electronic databases/websites (NCBI)
Introduction to PDB Protein Data Base To search for relationships between protein
BLAST Sequences http://www.ncbi.nlm.nih.gov/
III. Biomanufacturing-Products and Applications of Modern Biotechnology_
A. Assays & Assay Development-- Identifying a Potential Biotechnology Product 6
Sources of Potential Products Biotech Online-Amazon Hide and Seek
Introduciton to the Use of Assays Lab-How Do You Know You Have Amylase?
Looking for New Products in Nature Lab-Assaying for Starch and Sugar/protein
B. Bringing the Products of Biotechnology to Market
R & D for Potential Products
Upstream/Downstream
Clinical Testing FDA approval Create a Company & Product to bring to market
Chromatography Lab-Paper vs. Column Chromatography
Intro to Quality Assurance
C. Spectrophotometer 7
Using a Spectrophotometer to Detect Molecules
Acids/Bases/Buffers Lab-Calibrating and Using a pH Meter
Using the Spectrophotometer to Measure Protein Lab-Measuring the pH of Solutions
Concentration Lab-Determining the Concentration of solutions
D. Recombinant DNA & Gneetic Engineering 8
An Overview of Genetic Engineering Lab-Restriction Analysis with Lambda Phage
Plasmid Technology Lab-Transformation of E. coli with pGLO
Polymerase Chain Reaction PCR Lab PCR Crime Scene
Transforming Cells Lab-Growing & Monitoring Bacterial Cultures
Making Recombinant DNA
Fermentation Lab-Fermentation of Yeast
E. Bioethics
Cloning
Stem Cells http://learn.genetics.utah.edu.
Eugenics Web based exercise on Cloning
Gene Therapy Web-Eugenic Movement
ELSI Ethical; Legal and Social Implications
Human Genome Project
F. BIOINFORMATICS
Identify & Utilize electronic databases/websites (NCBI)
To search for relationships between protein
Sequences http://www.ncbi.nlm.nih.gov/
G. Biotechnology in Agriculture & Environment 12
Biological Weapons
Environmental spills: bacteria cleanup
GMOs Lab: Identifying GMO in food products
Bioethics: Monsanto vs. Farmers Web Search: debate pros & cons
IV Career Pathways in Biotechnology
A. Survey the Biotechnology Field Web-Search Activity & Presentation
Job Search Skills Web-Search Laws and Restrictions
Employability Skills Write a Resume
B. Develop Individual Career Plan Fill out Sample Applications
Prepare for Employment Write Cover Letter
Oral & Written Communication Skills Write a Letter of Recommendation
Mock Interviews
C. Quality Assurance
How to handle complaints; harassment; bioethics
D. Work-Based Learning
Final Project-Career Presentation
Prepare Pamphlet
Dress for Success
Chapter 1 I laboratory Manual
Table 1.1. Safety Hazards and Safety Equipment Chart
boratory 1c Cheese Production: The Evolution of Cheese-Making Technology
This activity was inspired by labs developed by Louann Carlmagno; formerly of Genencor International; Inc.
The cheese-making industry is huge and has a great number and variety of products (see Figure 1.6). Cheese-making is a good example of how biotechnology has improved an indus.trial process.
In the past; people made cheese simply by letting the naturally occurring bacteria in milk turn the milk sour. In. that process; the bacteria use milk sugar (lactose) as an .. energy source and produce lactic acid; a waste product. Lactic acid also causes the mixture to have a mild to slightly bitter taste. Along with other flavorful compounds; the lactic acid gives the cheese a characteristic flavor.
The milk bacteria produce special enzymes (proteins
; that speed reactions) that convert the lactose to lactic acid.
•Lactic acid has a low pH (the hydrogen ion concentration; or a measure of the acidity) and causes the milk protein; casein; to denature (unwind) and fall out of solution.
. Other enzymes; called proteases; may also act on casein.
; Proteases cleave proteins; such as casein; into smaller
; fragments that will also fall out of solution. The lumps of
'
Figu•re- . . ConSUiTier:S have many varieties-of cheese to choose from. Recently; biotechnology products that make cheese faster than older methods have improved the cheese­ making process.
Photo by author.
denatured casein are called curds. Curds are pressed
together to form cheese. The liquid remaining after cur­ dling is called whey.
Using early methods; cheese makers started new batch-
es with a small amount of cheese (containing the enzyme­
producing bacteria) they had saved from a batch of curdled milk that produced a good cheese. Although some cheese is still produced in this fashion; today; most commercially made cheese is produced in one of the four ways listed below. In each method; sterilized milk is used as a start­ ing reagent.
1. The milk may simply be left to age; exposed to air and naturally occurring bacteria (see Figure 1.7).
2. New batches of cheese are started with specific cultures of selected bacteria. These "known" bacteria also make the enzymes that curdle milk. Buttermilk has a good culture of Lactobacillus bacteria and can be used as a "starter" (see Figure 1.8).
3. New cultures may be started by the addition of purified enzymes; such as rennin; which is
retrieved from the cells lining the stomachs of calves (see Figure 1.9). Rennin is a type of protease; and like other proteases; it cleaves the casein into small fragments that settle out
3
Introduction 10 Biotechnology Methodologies
whey­ the liquid left after curdling
Even "freshH milk has bacteria in it. The bacteria use the milk protein; casein; as food.
As the bacteria grow; they produce products that make the protein fa!! out of
solution •in lumps. The lumps are called "curds."
Figure 1.7. Milk Curdling Milk left exposed to air curdles because of bacterial enzyme activity.
Milk is heated until just before boiling and then cooled to about 30°C.
' The bacteria produce products that curdle
the milk into semi"sol1d form.
Yogurt; buttermilk; or cheese with active cultures of certain selected bacteria is stirred into the warm milk.
Figure 1.8. Milk Curdling with Starter Adding an existing culture of mi!k"curdling bacteria will speed curdling in a milk sample.
as curds. When calves nurse; their stomach cells produce rennin to digest the milk protein. To retrieve the calves' enzymes for commercial use; companies grind up the calf stomachs and purify the rennin enzyme from all of the other compounds made by the cells. For this reason 1 some vegetarians do not eat rennin cheeses. There are many rennin cheeses 1 including Asiago; most bries; most cheddars; and Roquefo1t.
4. New cultures may be started by adding purified enzymes produced by genetic engineering (see Figure 1.10). Scientists found the DNA code for the cheese-making enzymes produced by calves in regular cow cells. They cut out the cow's rennin cheese-making code (gene) and inserted it into fungus cells. Fungus cells then read the cow DNA and synthesized the
Chapter 1 \ Laboratory Manual
Calf stomach cells make an enzyme protein called rennin. Rennin
causes the milk protein; casein; to
fall out of solution to form curds.
Scientists can burst the ca!! stomach cells open and isolate
rennin to use in commercial settings.
Purified rennin enzyme can be added directly to milk.
•;';!i
Concentration and volume t'
of enzyme can be controlled. 4
!
Enzyme activity causes milk curdling. The curds are pressed into cheese.
Rennin can be Isolated from an the other proteins the stomach cells release.
Figure 1.9. Milk Curdling with Rennin The enzyme rennin is extracted from calf stomachs. Rennin curdles milk
by breaking bonds that hold the casein protein together.
Cows only produce rennin in stomach ce!ls; but every cow eel! contains the DNA code for rennin; whether the cell makes rennin or not.
fungus cells
J-:. '"r"enn'i'n
recombina.nt DNA
the
gene from
- \he cow
-' • Cow DNA is inserted
fungus DNA into fungus ONA.
"'recombinent DNA
mRNA for rennln is made from the
Purified chymosin causes
curds. Curds can be
pressed into cheese.
cheese made using a genetically engineered recombinant DNA product
milk.
I
!
'
t chymosin
cow rennin made ln fungus
cells; now called chymosin"
rennin gene.
;;
Figure 1.10. Milk Curdling with Chymosin The genetically engineered enzyme chymosin can be produced in fungi faster and more economically than in calves. Chymosin is a form of cow rennin produced in fungi.
rennin enzyme; which scientists called "chymosin." Then; cheese makers used the geneti­ cally engineered enzymes to speed curdling. Now; fungal cultures produce the curdling
enzymes in a faster; cheaper manner
and in larger amounts than can be produced inside
big; multicellular organisms; such as cows. Chymosin cheeses include Jack; mozzarella; and
most Swiss cheeses; plus many others.
'
lntroduclion to Biotechnoloov Methodologies
Scientists work to create new and improved versions of cheese-curdling enzymes; as well as to improve the yields and qualities of cheeses. Modern-day cheese makers want to produce large amounts of high-quality cheese in the most economical way.
Purpose
Determine which curdling agent produces cheese the fastest. Determine which curdling agent produces the most cheese. Examine numerical data to support predictions.
Examine variables that can lead to invalid experiments.
Hypothesis
Since chymosin is a product of scientific manipulation; it might be expected to produce the largest volume of cheese in the shortest amount of time.
Materials
Tubes; 15 mL; sterile Pipet; 10 mL
Pipet pump; green
Whole milk
Micropipet; P-1000
Micropipet tips for P-1000
Pipet; 1mL
Pipet pump; blue
Buttermilk
Rennin; bovine
Chymosin; recombinant rennin
Test tube racks for 15 mL tubes
Graduated cylinder; 25 mL Plastic funnels; short-stemmed Filter paper; 12.5 cm
Water bath; 37°C
Permanent lab marker pens
1. Using a 10-mL pipet and pipet pump; transfer exactly 7 mL of whole milk into a labeled; 15-mL conical tube.
2. Using a preset P-1000 micropipet or a 1-mL pipet and pump; add 0.25 mL (250 µL) of one of the four curdling agents to the 7 mL of milk. Use buttermilk; rennin; chymosin; or more
whole milk (negative control) as assigned by your supervisor.
3. Cap the tube and mix by gently inverting three times. Record this "initial time."
4. Place the milk-containing portion of the tube deep in your armpit; like a thermometer; and
incubate it there for at least 15 minutes.
5. Check for curdling every 5 minutes; recording the time to curdle
'
in minutes. To check for curdling; gently tilt the tube; being care­ ful to not break up any curds. Curds are large lumps of solidified milk. After 15 minutes; place the tube upright at room tempera­ ture and check for curdling every 15 minutes for 2 hours. If cur­ dling has not occurred within 2 hours; continue checking once every 4 hours. With the greatest accuracy possible; record the time; in minutes; until the milk curdles to the greatest extent.
6. If curdling has not occurred by the end of the laboratory peri­
od; bring the tube home (keep at room temperature) and back to class in 24 hours. Keep the tube upright so any curds fall to the bottom of the tube.
7. On your return to the lab; measure the volume of curds (solids) and whey (liquid) in the tube. You may be able to read the volume of each directly from the tube; although it may be difficult. Better yet; filter the curds as described below; using a "whey-o-meter" (see Figure 1.11).
8. On your return to the lab; pour the whey and curds mixture through a filter paper funnel into a 10-mL graduated cylinder
(a "whey-o-meter"). Determine the volume of whey collected in the graduated cylinder; using a pipet; if necessary; to measure
small amounts. By subtraction; determine the volume of curds. Can you suggest another method to determine the amount of curds produced in each treatment?
plastic funnel
cone curds and
whey
mixture
Whey
collects.
Figure 1.11. Whey-o-meter A whey-o-meter measures the amount of whey in a curdled milk sample. By subtracting the whey volume from the total volume of reagents used; you can estimate the volume of curds.
-
Chapter 1 I Laboratory Manoni
Table 1.2. The Effects of Cheese-Curdling Agents on Curdling Tiffie and Volume
buttermilk
rennin
chymosin
milk (control)
9. In your notebook; in a data table similar to Table 1.2; record the data for your sample plus one each of the other variable groups. This will give you data for one experimental trial of each curdling agent. Record the name of the person from whom you obtained data.
How well do these single trials of the experiment support' the original hypothesis? Explain.
Using Microsoft® Excel®; the lab supervisor or a student colleague will enter each individual's data into a class data table showing multiple replications of the experiment. Averages for each variable group should also be recorded in this data table. Averaged data are the best answer to an experimental question. Can you explain why?
Data Analysis
Using Microsoft® Excel® or a piece of graph paper; produce two graphs: one showing the aver­ age time to curdling for each enzyme treatment and one showing the average volume of curds produced by each enzyme treatment. Use the Microsoft® Excel® tutorial; "How to create a
chart; u if necessary.
Imagine you are an employee at a cheese company and you must summarize the results of your experiments and give your supervisor the "best" answers to the scientific questions asked. Write a conclusion that thoroughly reports and analyzes the experimental data. Following the "REE; PE; PA" method of writing the conclusion ensures a thorough discussion of the experimental results. REE stands for "results" with "evidence" and "explanation;" PE stands for "possible errors;'1 and PA represents "practical applications."
In the first paragraph of your conclusion; describe the results of the experiment (answer to the purpose question); including evidence and explanations for your findings. Discuss how well the results support the hypothesis. (This is "REE.")
In the second paragraph; identify sources of errors in the procedure that may lead to varia­ tions in results or invalid data. Identify the error and explain what might happen as a result of the error. (This is "PE.")
In the third paragraph; make a recommendation to the company supervisor. Identify which curdling agent should be used for production or the target of continued testing. Include a dis­ cussion of any adjustments in the procedures that you think may improve cheese production. Include a proposal for the next set of experiments. (This is "PA.")
Witnessing
In the biotechnology industry; the work of others is reviewed and "OK'd" by peers. This is called "witnessing." When you witness data and review a concluding statement; check for the following:
• accuracy of statements (and that they make sense)
• completeness (REE; PE; PA)
• evidence (numerical data with units of measurement)
• grammar and spelling errors
lnlroduction to Biotechnology Methodologies
Make corrections and suggestions right on the page in ink.
• For corrections; draw a single line through the error; correct it; and initial it.
• Write your suggestion for the correction in the margin and draw an arrow to where it should be placed. Then initial and date your entry.
Witness a colleague's conclusion. When you complete the witnessing; write; "Witnessed by" at the end of the conclusion; and then write your full name and the date.
The curdling agent experiment has so many factors or variables that can impact the results. To have confidence in the results; you will need a thorough examination of all factors to be controlled.
1. In your notebook; make a chart similar to Table 1.3 that has at least eight rows and four columns. You will use the table to analyze the variables in this experiment that need to be controlled if a technician were to have confidence in the results.
2. In this experiment; each curdling agent is tested multiple times and an average result is determined. Look at the class data. Does it appear that the number of replications for each curdling agent experiment was sufficient? Yes or no? Explain your answer.
3. Do you think that the whey-o-meter instrument was adequate for an accurate determination of whey volume and; indirectly; the curd volume? Why or why not? If yes; explain why. If no; propose a better system to determine the volume of curds or the volume of whey.
Table 1.3. Analysis of Variable(s) in Curdling Experiment
temperature Hands have different temperatures. Place all tubes in a 37°( water hath. 8
Not as important to see difference in buttermilk or negative control.
Very important to see any difference between chymosin and rennin.
Samples are held for different time periods.
Higher temperatures may give fast reactions.
Have timer set to ring every 5 minutes.
Lift and invert one time; then return to hath.
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
- CTE
- Career and technical education
- LADV
- Advanced science
- LINT
- Integrated science