EDVOTEK Workshops offered Thurs. Nov. 14 at the CAST24 Conference in San Antonio, TX. 1. Forensic Escape Room: Design Your Own Biotech Adventure 2. Sweet Science: Exploring Complex Mixtures with Biotechnology 3. BioArt: Exploring STEAM with Transformation 4. Introducing Your Students to CRISPR with Sickle Cell Gene Editing 5. Biotechnology Basics: My First Electrophoresis
EDVOTEK® WORKSHOPS • CAST 24 San Antonio
TABLE OF CONTENTS
WORKSHOP
PAGE
01 Forensic Escape Room: Design Your Own Biotech Adventure 02 Sweet Science: Exploring Complex Mixtures with Biotechnology
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15 27 39 51
03 BioArt: Exploring STEAM with Transformation
04 Introducing Your Students to CRISPR with Sickle Cell Gene Editing
05 Biotechnology Basics: My First Electrophoresis
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Introduction Explore the world of forensic science with these fun and exciting escape room activities! Try forensic blood detection and agarose gel electrophoresis experiments, decipher clues, and solve puzzles. Learn to design your own escape room to have students unravel the evidence and free the innocent. Background Information Excerpts from EDVO-Kits 191 & 130 An abundance of material evidence can be left behind at the scene of a crime. This evidence can include blood on clothing, walls or floors, or even on the potential murder weapon. In some cases a few cells caught under the victim’s nails during a struggle can provide a wealth of information. Evidence can be obtained based on microscopic examination and biotechnological analysis, and then compared to a sample obtained from a person of interest who may have been at the site of the crime. Advances in molecular biology and genetics over the past 30 years have produced a variety of applications that have forever changed forensic science. Human tissue is made up of cells that contain DNA, which can be col- lected from evidence. When combined with the polymerase chain reaction (PCR) and DNA fingerprinting a very small amount of DNA from a biological sample can be analyzed. In many cases the crime can only be solved, and the criminals brought to justice, through the meticulous work of forensic scientists. DETECTION OF BLOOD SPATTERS: Presumptive & Confirmatory Tests The most common presumptive forensic blood test is the Kastle-Meyer test (Box 1). The Kastle-Meyer test uses a compound known as phenolphthalein (pr. fee-nawl-thal-een), which reacts with the iron carried by hemoglobin. First, presumptive blood is gathered on a cotton-tipped swab. The cellular membranes of cells on the swabs are then broken open (lysed) by applying a few drops of 95% ethanol. Phenolphthalin solution is then applied, followed quickly by hydrogen peroxide. If the cotton swab turns pink, it means that there was likely hemoglobin in the sample. BOX 1: Chemistry of the Kastle-Meyer Test The phenolphthalein (C 2 0H 16 O 4 ) used in the Kastle-Meyer test has been reduced, i.e. it has gained electrons, and is actually called phenolphthalin (C 2 0H 14 O 4 ) . The reaction in the Kastle-Meyer test is based on the reac- tion between the iron in hemoglobin and hydrogen peroxide (H 2 O 2 ). The iron in hemoglobin reduces (sup- plies electrons to) the H 2 O 2 , creating water (H 2 O). This reaction depletes the hemoglobin of electrons, which are in turn supplied by phenolphthalin. The oxidation, i.e. the release of electrons, of phenolphthalin turns it back into phenolphthalein, which has a characteristic pink color. Fe 4+ + C 2 0H 14 O 4 + H 2 O 2 → C 2 0H 16 O 4 + H 2 O + Fe 3+ Presumptive tests, such as the Kastle-Meyer, must be confirmed using a test that definitively detects blood. These are known as confirmatory tests. Confirmatory tests are often much more expensive and can take more time than presumptive tests. The most common confirmatory test for blood is the Rapid Stain Identification of Human Blood (RSID). The RSID works similarly to a pregnancy test. The sample is applied to the test strip, and antibodies that recognize blood proteins specifically bind to the sample. If the antibodies bind and the sample is positive for blood, a visible line is shown in the viewing window. Another confirmatory test for blood is ABO blood type testing. Testing for blood groups relies on the precipitation of an antigen-antibody complex, called agglutination. Only blood will produce this agglu- tination, which is why it is classified as a confirmatory blood test. In addition to being a confirmatory test, ABO blood typing is also a faster and more affordable identity test than other analysis techniques such as DNA fingerprinting. Indeed, forensic blood typing serves both as a confirmatory test and provides information about the suspect in the form of their blood type. Even though blood typing cannot point to a specific person as the criminal, it can point to a group of people that share the same blood type or eliminate suspects whose blood type does not match.
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
BLOOD TYPING Blood typing is an immensely important clinical procedure. It is one of the first procedures performed during blood transfusions and surgery, and is also important in forensic science. Blood typing is an example of an agglutination assay, the precipitation of antigens on red blood cells and antibodies in the blood. When both components are present at a similar concentration they are in a state known as
equivalence. In an equivalent state, neither the antibody nor the antigen is in excess, and the antigen-antibody complex forms large networks that precipitate out of solution. Impor- tantly, the precipitate is easy to detect by eye, making agglutination assays both easy and cost-effective to perform. The most common blood typing system relates to the presence of the A and B antigens on the red blood cells. This system, known as the ABO blood types, produces four
Antigen on Red Blood Cells
Antibody in Blood
Percentage of Population
Blood Type
A B AB O
A B A & B O
anti-B anti-A none anti-A & anti-B
42% 10% 4% 44%
Figure 1: Types of Blood in the Population
possible blood types: A, B, AB, and O (Figure 4). Individuals with only A antigens will have type A blood, while someone with B antigens has type B blood. The A and B antigens are co-dominant, so a person can have both antigens on their red blood cells, leading to the AB blood type. If an individual has neither A nor B antigens they have type O blood. A person with type A blood will recognize red blood cells with the A antigen as “self”. However, if that person gets a blood transfusion with type B blood, the new red blood cells will be recognized by the immune system as foreign and will cause an immune response. Antibodies targeting the B antigens (anti-B antibodies) will bind to the B antigen on the transfused cells and agglutinate. In many cases, this severe immune response can be deadly. Therefore, it is important for hospitals and clinics to maintain records of patient blood types. The same reaction that can lead to severe immune responses in a patient is used for clinical and foren- sic blood typing experiments. For example, type B blood can be easily recognized by the agglutination between the anti-B antibodies and the B antigen. When something that is suspected to be blood is found at a crime scene, detectives will work quickly to secure the evidence and send it to a forensic lab for testing. In the lab, forensic scientists will perform presumptive and confirmatory tests for blood, potentially recommending additional testing such as DNA profiling. DNA FINGERPRINTING In humans, DNA is packaged into 23 pairs of chromosomes that are inherited from an individual’s bio- logical parents. Although most of this genetic material is identical in every person, small differences, or “polymorphisms”, in the DNA sequence occur throughout the genome. For example, the simplest differ - ence is a Single Nucleotide Polymorphism (or SNP). Changes in the number and location of restriction enzyme sites result in Restriction Fragment Length Polymorphisms (or RFLPs). Short repetitive stretches of DNA at specific locations in the genome can vary in number to produce STRs (Short Tandem Repeats) and VNTRs (Variable Number of Tandem Repeats). Although most polymorphisms occur in non-coding regions of DNA, those that disrupt a gene can result in disease. Medical diagnostic tests can identify specific polymorphisms associated with disease. Analyzing several different polymorphisms within a person’s genome generates a unique DNA “finger - print”. DNA fingerprints can allow us to distinguish one individual from another. Because polymorphisms are inherited, DNA fingerprints can also be used to determine paternity/maternity (and other familial relationships). The best-known application of DNA fingerprinting is in forensic science. DNA fingerprint - ing techniques are utilized to interpret blood, tissue, or fluid evidence collected at accidents and crime scenes. After DNA is extracted from these samples, forensic scientists can develop a DNA fingerprint. The DNA fingerprint from a crime scene can then be compared to the DNA fingerprints of different suspects. A match provides strong evidence that the suspect was present at the crime scene.
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Early fingerprinting analysis involved restriction digestion of the isolated DNA. Following electrophoresis of the digested sample, the DNA is transferred to a nylon membrane during a process known as Southern blotting. Sequence-specific DNA probes are used to visualize the membrane-bound DNA. If the DNA is not digested by the restriction enzyme, the probes will only hybridize to a single DNA segment. If a restriction site occurs within this sequence, the probe will hybridize with multiple bands of DNA. VNTRs are identified when a probe labels DNA at a dissimilar molecular weight. Although RFLP analysis is very precise, it is time-consuming and requires large amounts of DNA. To address these problems, forensic scientists use the polymerase chain reaction (PCR) to produce DNA fingerprints. PCR allows researchers to quickly create many copies of a specific region of DNA in vitro (summarized in Figure 1-1). This technique requires 500-fold less DNA than traditional RFLP analysis and it can be performed in an afternoon. To perform PCR, purified double-stranded DNA is mixed with primers (short synthetic DNA molecules that target DNA for amplification), a thermostable DNA polymerase (Taq) and nucleotides. The mixture is heated to 94°C to denature the DNA duplex (i.e., unzip it into single strands).Next, the sample is then cooled to 45°C - 60°C, allowing the primers to base pair with the target DNA sequence (called “annealing”). Lastly, the temperature is raised to 72°C, the optimal temperature at which Taq polymerase will extend the primer to synthesize a new strand of DNA. Each “PCR cycle” (denaturation, annealing, extension) doubles the amount of the target DNA in less than five minutes. In order to produce enough DNA for analysis, twenty to forty cycles may be required. To simplify this process, a specialized machine, called a “thermal cycler” or a “PCR machine”, was created to rapidly heat and cool the samples. ESCAPE ROOM SCENARIO Monica Homewood has been arrested as the main suspect in the murder of her husband, Bryce Home- wood. She has been questioned multiple times and continues to plead innocent to the crime. Monica’s lawyer is trying to prove her innocence and has convinced the Charleston Police Department to further investigate the crime scene. You are the best forensic analyst that money can buy in Charleston. Your job is to determine if the evidence collected at the crime scene contains blood, what blood type the blood evidence is, and to use DNA fingerprinting to determine who committed the crime. Will your forensics skill help prove Monica’s innocence? The following information and evidence are required for your forensic assessment of the crime.
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
OFFICIAL POLICE REPORT
RESPONDING OFFICER: Caitlin Marlow
DETAILS OF EVENT: On Saturday night, police dispatch received a distressed phone call from a woman in her mid-30’s, claiming to have returned home to find her husband dead in their kitchen. Dispatch sent Caitlin Mar - low, the local police sheriff, to the crime scene in a small suburban condo in Charleston, South Caro - lina. Upon arriving at the condo, Sheriff Marlow found a deceased man with a traumatic head injury, including a deep laceration, in the kitchen. Marlow and her crime scene investigators collected the evidence from the scene to be analyzed by the crime scene team. Upon initial inspection of the scene, the following primary evidence was collect- ed: glass shards (from a broken iced tea pitcher) with what appears to be blood splatter on the kitchen table (Sample 1), a knife near the table with apparent blood on it (Sample 2), potential blood spatter on the hardwood floor near the kitchen table (Sample 3), and a tall glass with women’s lipstick on it. Later, the coroner was able to identify the body as Bryce Homewood. The cause of death was de - termined as a traumatic head injury. Glass shards in the head wound matched the broken glass from the iced tea pitcher found at the crime scene. A mysterious note with unintelligible text was found in the victim’s pocket. Marlow and her team of detectives questioned the neighbors in the condo complex. They deter- mined Monica Homewood, Deena Granville, and Alex Johnson to be the three main suspects of this crime. According to eyewitness reports, both Monica and Deena were seen at the scene of the crime one (1) hour before the call to the police dispatch. Neighbors also noted that a local real estate agent, Alex Johnson, was seen talking with Mr. Homewood at the condo the night before. THE VICTIM: • Bryce Homewood , aged 32, was a graphic designer for an advertising agency. He was married to Monica Homewood. He was known for his sharp wit and humor, though sometimes he veered into the realm of abuse and/or harassment when angry. SUSPECTS: • Monica Homewood , 33, works as a pediatric nurse at a local children's hospital. She was the distressed woman from the police dispatch phone call. She is also married to the victim. Most of the neighbors complained about their constant arguments. Their fights often disrupted the peaceful condo building. When questioned about her whereabouts during the crime, Monica claimed to have gone to an urgent care to take care of a deep wound she claims she obtained accidentally while cooking dinner with Bryce. It was confirmed by the local urgent care that Monica was treated for a knife wound. • Deena Granville , 34, is the neighbor and HOA president. She works as a marketing manager for a fashion retail company. She is known for being a stickler on the HOA rules and ever since the Home- woods moved in, there have only been fights between Bryce and Deena. Neighbors also noted that Bryce was also constantly fighting with Deena over excessive property management fees. The meeting notes from the HOA meetings prove that the two have a history of be- ing verbally aggressive towards each other. • Alex Johnson , 38 - Real Estate Agent. Alex Johnson has ongoing disputes with Bryce over a contest- ed condo sale. At the time of the crime, he claimed to be attending a local real estate conference which investigators have yet to confirm. Neighbors noted Alex had a strained relationship with the victim due to their frequent clashes over business deals, but he always maintained a professional demeanor in their interactions. SAMPLES PENDING FORENSIC ANALYSIS: Potential blood evidence found at the crime scene. Tests to be performed include Kastle-Meyer testing, Blood Group Typing and DNA Fingerprinting.
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Module I: Kastle-Maeyer Test
The first steps of your forensic analysis will use the Kastle-Meyer test as a presumptive test for blood. You will test the three crime scene samples to see if they are positive or negative for the presence of blood.
25 µL evidence
50 µL Phenolphthalin solution
50 µL Hydrogen peroxide solution
50 µL ethanol
1. Use a transfer pipet to ADD 1 drop (or 25 µL) of blood evidence to the swab. NOTE: Using more than one drop (25 µL) of evidence may affect the results.
2. Use a transfer pipet to ADD 2 drops (or 50 µL) of 95% ethanol to the swab. NOTE any color change. PLACE the pipet and remaining ethanol to the side for testing additional samples. 3. Use a new pipet to ADD 2 drops (or 50 µL) of the phenolphthalin solution to the swab. NOTE any color change. No color change is expected even if blood is present. PLACE the pipet and remaining phenolphthalin to the side for testing additional samples.
IMPORTANT For steps 2-4: When adding the detection reagents, hold the evidence swab at a steep angle, with the crime scene sample at the bottom.
4. Use a new pipet to ADD 2 drops (or 50 µL) of hydrogen peroxide solution “H2O2” to the swab. NOTE any color change. A pink color is expected after several seconds if blood is present. RECORD your results in the chart below.
REPEAT steps 1-4 for each blood evidence sample.
5.
Crime Scene Sample #
Positive or Negative for Blood
CS1
CS2
CS3
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Module II: Blood Type Test
Now that some samples from the crime scene have been identified as blood using the Kastle-Meyer test, it is necessary to confirm the blood type of the samples. In this module, you will perform ABO testing on the blood samples from the crime scene. This will allow us to determine which crime scene blood samples are from the victim, and which samples were from the suspects. NOTE: Bryce is blood type O, Deena is blood type B, Alex is blood type A, and Monica is blood type B.
1. PLACE a microtiter plate piece as shown below. Using a permanent marker, label the plate. The column labels will include the four control blood types (A, B, AB, O) and any Module I crime scene samples that test positive for blood. The row labels will be Anti-A and Anti-B.
A B AB O
Anti A
Anti B
2. ADD 50 μL or two drops of each control blood type sample into each of the two corre - sponding wells. For example, control blood type A goes into the two wells under the letter “A”. Repeat the same procedure for the control blood types B, AB, O, and the two positive blood samples from the crime scene. Each well requires 50 μL. 3. Use a new pipette tip to ADD 50 μL or 2 drops of Anti-A serum into each of the wells in row #1. The same tip or transfer pipet can be used for all samples in row #1. 4. Use a new pipette tip to ADD 50 μL or 2 drops of Anti-B serum into each of the wells in row #2. The same tip or transfer pipet can be used for all samples in row #2. 5. Firmly HOLD the plate piece on the lab bench as shown. Very gently TAP the side of the plate with your finger near the positive crime scene samples to MIX for approximately 20- 30 seconds. Do this VERY CAREFULLY to avoid spilling any of the samples from the wells. PROCEED to the next step. 6. To visualize the results, PLACE the microtiter plate on a white light transilluminator, or use a bright flashlight held above the plate. COMPARE the crime scene evidence with the control blood samples. RECORD your results.
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Module III: DNA Fingerprinting After using blood typing to determine which samples are from the suspect, you must use DNA fingerprinting to find out who the blood came from. You send the suspects’ blood samples as well as the blood collected from the knife to an external DNA lab. Here they will extract DNA from each sample and amplify it by PCR. In this module, you will use gel electrophoresis to analyze the PCR samples obtained from the suspects and compare them to the blood found at the scene.
1:00
Concentrated buffer
Distilled water
Agarose
60°C
Flask
Caution! Flask will be HOT!
WAIT
POUR
60°C
ADD diluted SYBR Safe
60°C
CASTING THE AGAROSE GEL 1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).
REMINDER: This kit requires 0.8% agarose gels cast with at least 4 wells.
2. MIX agarose powder with 1X buffer in a 250 mL flask (see Table A). 3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water). 4. COOL agarose to 60 °C with careful swirling to promote even dissipation of heat. 5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch. 6. Before casting the gel, ADD diluted SYBR® Safe to the molten agarose and swirl to mix (see Table A). 7. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies. 8. REMOVE end caps and comb.
Take particular care when re- moving the comb to prevent damage to the wells.
Individual 0.8% UltraSpec-Agarose™ with SYBR ® Stain
7 x 7 cm
0.6 mL
29.4 mL
0.24 g
30 mL
30 µL
10 x 7 cm*
0.9 mL
44.1 mL
0.36 g
45 mL
45 µL
14 x 7 cm
1.2 mL
58.8 mL
0.48 g
60 mL
60 µL
* Recommended gel volume for the EDGE™ Integrated Electrophoresis System.
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Module III: DNA Fingerprinting, continued
RUNNING THE GEL 9.
REMINDER: Before loading the samples, make sure the gel is properly oriented in the
PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer (See Table B for recommended volumes). The gel should be completely submerged. 10. PUNCTURE the foil overlay of the QuickStrip™ with a pipet tip. LOAD the entire sample (35 µL) into the well as indicated by the Gel Loading table.
apparatus chamber.
11. PLACE safety cover. CHECK that the gel is properly oriented. Re- member, the DNA samples will migrate toward the positive (red) electrode. 12. CONNECT leads to the power source and PERFORM electrophoresis. (See Table C for time and voltage guidelines.) 13. After electrophoresis is complete, REMOVE the gel and casting tray from the electro- phoresis chamber.
GEL LOADING TABLE
VISUALIZING the SYBR® GEL SLIDE gel off the casting tray onto the viewing surface of the transilluminator. TURN the unit on. DNA should appear as bright green bands on a dark background. PHOTOGRAPH results.
SAMPLE A B C D
LANE 1 2 3 4
SAMPLE NAME DNA standard marker Crime scene sample Deena PCR Reaction Monica PCR Reaction
Time and Voltage Guidelines (0.8% Agarose Gel)
1x Electrophoresis Buffer (Chamber Buffer)
EDGE™
M12 & M36
Volts
Min/Max (minutes)
Min/Max (minutes)
EDGE™
150 mL
3 mL
147 mL
150 125 100
10/20
20/35 30/45 40/60
M12
400 mL
8 mL
392 mL
N/A
M36
1000 mL
20 mL
980 mL
15/25
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Experimental Results and Analysis
MODULE I ANALYSIS
Negative Sample
Positive Sample
CS1 was negative and did not produce any color change after adding H 2 O 2 , while CS2 and CS3 turned bright pink and were posi- tive. Therefore, CS2 and CS3 should continue on for confirmatory testing.
MODULE II ANALYSIS
The crime scene sample CS2 is type O and crime scene sample CS3 is type B blood. Both Deena and Monica are type B. While blood typing can narrow down suspects, DNA testing would have to be
performed to conclusively say if the blood belongs to either of the two suspects. Therefore, PCR samples from crime scene sample CS3 should be DNA tested alongside PCR samples from Monica and Deena.
MODULE III ANALYSIS
The DNA standards in Lane 1 make it possible to measure the DNA bands obtained from the PCR reactions. The results of this analysis indicates an identical pattern in Lanes 2 and 4. This is strong evidence that the crime scene DNA matches the sample from Monica. In criminal investigations, several known variable regions in DNA are analyzed to match crime scene and suspect DNAs.
TUBE A B C D
LANE 1 2 3 4
SAMPLE NAME DNA standard marker Crime scene sample Deena PCR Reaction Monica PCR Reaction
MOLECULAR WEIGHTS 6751, 3652, 2827, 1568, 1118, 825, 630 3000, 1282 3000 3000, 1282
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
Related Products Forensic Escape Room: Design Your Own Biotech Adventure For 10 groups. Explore the world of forensic science with this fun and exciting crime scene escape room! In this investigation, students decipher clues, solve puzzles, and unravel the evidence to free the innocent. Hands-on techniques include forensic blood detection, blood typing, and DNA fingerprinting. Comprehensive instructions on how to set up an escape room are included. Cat. #190 DNA Fingerprinting by PCR Amplification For 8 gels. Forensic DNA fingerprinting has become a universally accepted crime-fighting tool. Recent advances use the polymerase chain reaction (PCR) to amplify human DNA obtained from crime scenes. This experiment, based on a crime scene scenario, has an inquiry-based component. Cat. #130 Forensics Enhancement Techniques For 10 groups. Trace amounts of blood are often sufficient to identify the individual responsible for any number of crimes, including murder, burglary, or assault. Enhancement procedures can make a small stain of body fluid or tissue visible to the naked eye. In this experiment, students will act as detectives follow- ing the aftermath of a drug bust involving gang warfare over territory. Reagents that are routinely used as a first screen will be utilized to detect simulated blood and DNA. In addition, biological materials will be recovered from splatters, blood trajectory, and small droplets of simulated human materials. Cat. #194
Forensic Toxicology For 10 groups. In today’s forensic science labora- tory, toxicologists identify drugs and toxins in
samples collected from crime scenes, victims, and potential suspects. If present, the toxicologist also determines whether the drug or toxin contributed to a person’s behavioral changes or death. In this forensic science experiment, students will use the Enzyme Linked Immunosorbent Assay (ELISA) to analyze simulated crime scene samples for the presence of drugs. Cat. #195
Whose Fingerprints Were Left Behind? For 10 groups. After a crime has been committed, the evidence left behind can identify a potential culprit, although a single piece of evidence is not usually enough to convict someone. Even in this age of DNA, fingerprints and blood stains are still important at helping to identify a criminal. In this experiment your stu- dents will learn to detect and analyze fingerprints and then use these techniques to solve a classroom crime . Cat. #S-91
Details for all these products and MORE can be found on our website!
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01 - Forensic Escape Room: Design Your Own Biotech Adventure
RELATED PRODUCTS
EDGE™ Integrated Electrophoresis System Runs one 10 x 7 cm gel Cat# 500
M12 Complete™ Electrophoresis Package For 1 or 2 Lab Groups Cat# 502-504
DuoSource™ 100/150 V, for 1 or 2 Units Cat# 509
QuadraSource™ 10-300 V, for 1 or 4 Units Cat# 5010-Q
White Light LED Transilluminator Cat# 552
EDVOTEK® Variable Micropipette 5-50 µL Micropipette Cat. # 590
TruBlu™ Jr Blue Light Transilluminator Cat# 555
Long Wave UV Light Cat# 969
Fixed Volume MiniPipette™ 35 µL MiniPipet™ Cat. # 587-2
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
Introduction
In this experiment, students will investigate how gel electrophoresis unlocks the color code by investigating food dyes used to make colorful candies.
Background Information Color is an integral aspect of our culture. Companies have long been using color additives in a variety of products, including candies, shampoos, perfumes, drinks, etc. All the colors that we see are a mixture of the three primary colors: red, green, and blue. That these three simple colors can be combined to form the myriad of colors that we see everyday makes them a great example of how complex things, such as colors, can be broken down into their individual components. In the classroom, a great way to break down colors is by examining color additives in food. The color of food has always been important for us. The early Romans believed that people not only eat with their palate, but also “eat with their eyes”. For centuring, humans have used dyes from natural ingredients to add color to food, drink, clothing, and other products. These days, manufacturers add colors to food to offset color loss due to product exposure to various environmental conditions and to make them look more attractive to consumers. As the use of color additives to food continues to grow, concerns regarding the addition of food colors to products also emerge. The seven more commonly used food dyes in the United States are shown in Table 2-1. However, the FDA and independent scientists are looking into whether or not certain dyes, such as yellow 5 and red 40, are linked to hyperactivity or allergic reactions.
Figure 2-1: Example of Candy Ingredient Label
AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis is widely used to separate molecules based upon charge, size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and pro- teins. Agarose gel electrophoresis possesses great resolving power, yet is relatively simple and straightforward to perform. The gel is made by dissolving agarose powder in the electropho- resis buffer. The solution is boiled to dissolve the agarose and then cooled to approximately 60º C and poured into a gel tray where it solidifies. The tray is submerged in a buffer-filled electrophoresis apparatus, which contains electrodes. Samples are prepared for electrophoresis by mixing them with glycerol or sucrose to give the mixture higher density. This makes the samples denser than the electrophoresis buffer. These
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
samples can then be loaded with a micropipet or transfer pipet into wells that were created in the gel by a template during casting. The dense samples sink through the buffer and remain in the wells. A direct current power supply is connected to the electrophoresis apparatus and current is applied. Charged molecules in the sample enter the gel matrix. Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode (cathode). Within a range, the higher the applied voltage, the faster the samples migrate. The buffer serves as a conductor of electricity and to control the pH. The pH is important to the charge and stability of biological molecules. Agarose is a polysaccharide derived from agar. In this experiment, UltraSpec-Agarose™, a mixture of agarose and hydrocolloids which renders the gel to be both clear and resilient, is used. At first glance, an agarose gel appears to be a solid at room temperature. However, on the molecular level, the gel contains microscopic pores which act as a molecular sieve, allowing the different molecules to pass through. Food dyes are composed of ions. When these charged ions are subjected to an electric field, the molecules will migrate toward the electrode of opposite charge. Positively charged mol- ecules will migrate toward the negative electrode, while those with a negative charge will move toward the positive electrode. Small dye fragments move through these holes easily, but large dye fragments have a more difficult time squeezing through the tunnels. Factors such as charge, size and shape, together with buffer conditions, gel concentrations and voltage, affects the mobility of molecules in gels. Because molecules with dissimilar sizes travel at different speeds, they become separated and form discrete “bands” within the gel. After the current is stopped, the bands can be visualized (Figure 2-2). In this experiment, students will extract several different dyes from food source. The dyes will then be analyzed using agarose gel electrophoresis and their rate of migration will be observed and measured.
( - )
Figure 2-2: Overview of agarose gel electrophoresis.
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
TABLE 2-1 – SEVEN ARTIFICIAL COLORS APPROVED BY THE FDA FOR COLORING FOOD
Abbreviation
Name
Shade
Structure
Brilliant Blue
Blue
Blue 1
Blue 2
Indigotine
Indigo
Green 3
Fast Green
Turquoise
Red 3
Erythrosine
Pink
Red 40
Allura Red
Red
Yellow 5
Tartazine
Yellow
Yellow 6
Sunset Yellow
Orange
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Experiment Overview
1 Prepare dye samples by extracting food dyes from candies
2
Prepare agarose gel in casting tray.
3
Remove end caps & comb, then submerge gel under buffer in electrophoresis chamber.
4
Load each dye sample in consecutive wells.
5
Attach safety cover & connect leads to power source to conduct electrophoresis.
After electrophoresis, transfer gel for visualization.
( - )
6
Analysis on white light source.
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
Module I: Extraction of Food Dyes from Candy
T.C. yell
250 µL Dye Extraction Buffer
T.C. blue
T.C. red
T.C.
T.C. pur
T.C. gre
SWIRL
RINSE your cup. REPEAT steps 2-6.
T.C. yell
T.C. red
T.C. pur
T.C. red
T.C. gre
T.C. blue
T.C.
We recommend using brightly-colored candies M&M’s®, Skittles®, jelly beans, & gum balls. 1. LABEL five microcentrifuge tubes with your initials and the colors of the candy you will be investigating. 2. LABEL the provided cup with your initials. ADD one candy to the cup 3. ADD 250 µL of Dye Extraction Buffer to the cup containing the candy. 4. SWIRL the candy gently in the Dye Extraction Buffer to dissolve the color coating until the white layer of the candy is exposed. 5. REMOVE the candy from the cup. 6. TRANSFER the dissolved color solution into the appropriately labeled microcentri- fuge tube. 7. RINSE the cup. REPEAT steps 2-6 with the remaining 4 candies. 8. PLACE the tubes on lab bench. PROCEED to Module II: Separation of Food Dyes by Agarose Gel Electrophoresis. OPTIONAL STOPPING POINT Dye samples may be stored in the refrigerator for up to 24 hours before performing electrophoresis.
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Module II: Agarose Gel Electrophoresis
Concentrated buffer
Distilled water
Agarose
60°C
Flask
Caution! Flask will be HOT!
WAIT
POUR
60°C
CASTING THE AGAROSE GEL 1. DILUTE concentrated 50X Electrophoresis buffer with distilled water (refer to Table A for correct volumes depending on the size of your gel casting tray). 2. MIX agarose powder with buffer solution in a 250 mL flask (refer to Table A). 3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water). 4. COOL agarose to 60°C with careful swirling to promote even dissipation of heat. 5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch. 6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transpar - ent as it solidifies. 7. REMOVE end caps and comb. Take particular care when removing the comb to pre- vent damage to the wells.
Individual 0.8% UltraSpec-Agarose™ Gels
7 x 7 cm
0.6 mL
29.4 mL
0.24 g
30 mL
*Recommended gel volume for the EDGE™ Integrated Electrophoresis System (Cat. #500).
10 x 7 cm*
0.9 mL
44.1 mL
0.36 g
45 mL
14 x 7 cm
1.2 mL
58.8 mL
0.48 g
60 mL
* Recommended gel volume for the EDGE™ Integrated Electrophoresis System.
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
Module II: Agarose Gel Electrophoresis, continued
RUNNING THE GEL 8. PLACE the gel (still on the tray) into the electrophoresis chamber. COVER the gel with 1X Electrophoresis Buffer (See Table B for recommended volumes). The gel should be completely submerged. 9. PUNCTURE the foil overlay of the Quick-
TABLE 2: GEL LOADING
Strip™ with a pipet tip. LOAD the entire sample (35 µL) into the well in the order indicated by Table 2, at right. 10. PLACE safety cover on the unit. CHECK that the gel is properly oriented. Re- member, the DNA samples will migrate toward the positive (red) electrode. 11. CONNECT leads to the power source and PERFORM electrophoresis (See
Candy Color
Lane
Standard Dye Marker
1 2 3 4 5 6
Table C for time and voltage guidelines). Allow the tracking dye to migrate at least 3 cm from the wells. 12. After electrophoresis is complete, RE- MOVE the gel and casting tray from the electrophoresis chamber and VISUALIZE the results. No staining is necessary.
Time and Voltage Guidelines (0.8% Agarose Gel)
1x Electrophoresis Buffer (Chamber Buffer)
EDGE™
M12 & M36
Volts
Min/Max (minutes)
Min/Max (minutes)
EDGE™
150 mL
3 mL
147 mL
150 125 100
10/20
20/35 30/45 40/60
M12
400 mL
8 mL
392 mL
N/A
M36
1000 mL
20 mL
980 mL
15/25
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
Module III: STEM-Based Data Analysis of Food Dyes Using a Standard Curve
Agarose gel electrophoresis separates biomolecules into discrete bands, each comprising molecules of the same size. How can these results be used to deter- mine the lengths of different fragments? Remember, as the length of a biomolecule increases, the distance to which the molecule can migrate decreases because large molecules cannot pass through the channels in the gel with ease. Therefore, the mi- gration rate is inversely proportional to the length of the molecules—more specifi - cally, to the log 10 of molecule's size. To illustrate this, we ran a sample that contains bands of known lengths called a “standard”. We will measure the distance that each of these bands traveled to create a graph, known as a “standard curve”, which can then be used to extrapolate the size of unknown molecule(s). 1. Measure and Record Migration Distances Measure the distance traveled by each Standard Dye Molecule from the lower edge of the sample well to the lower end of each band. Record the distance in centime- ters (to the nearest millimeter) in your notebook. Repeat this for each dye fragment in the standard. Measure and record the migration distances of each of the fragments in the un- known samples in the same way you measured the standard bands.
A B C
Figure 2-3: Measure distance
migrated from the lower edge of the well to the lower edge of each band.
2. Generate a Standard Curve. Because migration rate is inversely proportional to the log 10 of band length, plotting the data as a semi-log plot will produce a straight line and allow us to analyze an exponential range of fragment sizes. You will notice that the vertical axis of the semi-log plot appears atypical at first; the distance between numbers shrinks as the axis progresses from 1 to 9. This is because the axis represents a loga- rithmic scale. The first cycle on the y-axis corresponds to lengths from 100-1,000 base pairs, the second cycle measures 1,000-10,000 base pairs, and so on. To create a standard curve on the semi-log paper, plot the distance each Standard Dye Molecule migrated on the x-axis (in mm) versus its size on the y-axis (in base pairs). Be sure to label the axes!
QUICK REFERENCE: The Standard dyes have the fol- lowing base pair equivalents.
Blue Red
5000 3000 1000
Purple Orange
500
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
8,000 10,000 9,000
7,000 6,000 5,000 4,000
3,000
2,000
1,000
800 700 600 500 400 900
300
200
100
80 70 60 50 40 90
30
20
10
3 cm
2 cm
4 cm
5 cm
1 cm
6 cm
X-axis: Migration distance (cm)
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
Experimental Results and Analysis
1 2 3 4 5 6
1 2 3 4 5 6
NOTE: In the idealized schematic, the relative posi- tions of dye fragments are shown but are not depicted to scale. No positively charged dyes are shown.
Lane
Sample
1 2 3 4 5 6
Standard Dye Marker
Dye extracted from Candy #1 Dye extracted from Candy #2 Dye extracted from Candy #3 Dye extracted from Candy #4 Dye extracted from Candy #5
QUICK REFERENCE: Standard Dye marker sizes - length is expressed in base pairs.
5000, 3000, 1000, 500
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02 - Sweet Science: Exploring Complex Mixtures with Biotechnology
Related Products
M12 Complete™ Electrophoresis Package For 1 or 2 Lab Groups Cat# 502-504
M36 HexaGel™ For 1 to 6 Lab Groups Cat# 515
QuadraSource™ 10-300 V, for 1 or 4 Units Cat# 5010-Q
DuoSource™ 100/150 V, for 1 or 2 Units Cat# 509
White Light LED Transilluminator Cat# 552
Fixed Volume MiniPipet™ 35 µL MiniPipet™ Cat. # 587-2
EDVOTEK® Variable Micropipette 5-50 µL Micropipette Cat. # 590
For 10 Groups. NGSS-aligned with MS-PS1. Investigate how agarose gel electrophoresis unlocks the color code used by food scientists to make colorful candies. Students will extract color activities from common candies and separate the dyes on agarose gel electrophoresis. A fun lab exten- sion involves the use of candy to build a DNA model. Cat. #S-47 Linking Food Science to Biotechnology: Unlock the Color of Candies
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03 - BioArt: Exploring Transformation with STEAM
03
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03 - BioArt: Exploring Transformation with STEAM
Introduction Are transformations giving you trouble? Then this is the workshop for you! In this workshop, you will transform E. coli with plasmids that express brightly colored rainbow proteins. They can even glow fluorescent green with GFP! We’ll share tips and tricks along the way to ensure experimental success.
Table of Contents
Page
Background Information Experimental Procedure
3 5 8 9
Experiment Results and Analysis
7-8
Incorporating STEAM into Your Transformation Experiment
Troubleshooting Guide Workshop Products
10
Related Products
11-12
Online Resources We’ve created FREE Quick Guide manuals, for you to download from our website: www.edvotek.com/guides-lesson-plans We also have many Instructional Videos on Youtube that show step-by-step procedures: www.youtube.com/EdvotekInc
We hope you take advantage of these resources and enjoy teaching and learning with EDVOTEK®!
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03 - BioArt: Exploring Transformation with STEAM
Background Information DNA CAN BE TRANSFERRED BETWEEN BACTERIA In nature, DNA is transferred between bacteria using two main methods— transformation and conjugation. In transformation, a bacterium takes up exogenous DNA from the surrounding environment (Figure 1). In contrast, conjugation relies upon direct contact between two bacterial cells. A piece of DNA is copied in one cell (the donor) and then is transferred into the other (recipient) cell. In both cases, the bacteria have acquired new genetic information that is both stable and heritable. Frederick Griffith first discovered transformation in 1928 when he observed that living cultures of a normally non-pathogenic strain of Streptococ- cus pneumonia were able to kill mice, but only after being mixed with a heat-killed pathogenic
Bacterial Cell
Plasmid
Transformed Cell
Figure 1: Bacterial Transformation
strain. Because the non-pathogenic strain had been “transformed” into a pathogenic strain, he named this transfer of virulence “transformation”. In 1944, Oswald Avery and his colleagues purified DNA, RNA and protein from a virulent strain of S. pneumonia to determine which was responsible for transformation. Each component was mixed each with a non-pathogenic strain of bacteria. Only those recipient cells exposed to DNA became pathogenic. These transformation experiments not only revealed how this virulence is transferred but also led to the recognition of DNA as the genetic material. The exact mode of transformation can differ between bacteria species. For example, Hae- mophilus influenzae uses membrane-bound vesicles to capture double-stranded DNA from the environment. In contrast, S. pneumoniae expresses competency factors that allow the cells to take in single-stranded DNA molecules. In the laboratory, scientists can induce cells—even those that are not naturally competent—to take up DNA and become transformed. To accomplish this, DNA is added to the cells in the presence of specific chemicals (like calcium, rubidium, or magnesium chloride), and the suspension is “heat shocked”—moved quickly between widely dif- ferent temperatures. It is believed that a combination of chemical ions and the rapid change in temperature alters the permeability of the cell wall and membrane, allowing the DNA molecules to enter the cell. Today, many molecular biologists use transformation of Escherichia coli in their experiments, even though it is not normally capable of transforming in nature. GENETIC ENGINEERING USING RECOMBINANT DNA TECHNOLOGY Many bacteria possess extra, non-essential genes on small circular pieces of double-stranded DNA in addition to their chromosomal DNA. These pieces of DNA, called plasmids, allow bacteria to exchange beneficial genes. For example, the gene that codes for ß-lactamase, an enzyme that provides antibiotic resistance, can be carried between bacteria on plasmids. Transformed cells secrete ß-lactamase into the surrounding medium, where it degrades the antibiotic ampicillin, which inhibits cell growth by interfering with cell wall synthesis. Thus, bacteria expressing this gene can grow in the presence of ampicillin. Furthermore, small “satellite” colonies of untrans- formed cells may also grow around transformed colonies because they are indirectly protected by ß-lactamase activity. Recombinant DNA technology has allowed scientists to link genes from different sources to bacterial plasmids (Figure 2). These specialized plasmids, called vectors, contain the following features:
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03 - BioArt: Exploring Transformation with STEAM
1.
Origin of Replication: a DNA sequence from which bacteria can initiate the copying of the plasmid. Multiple Cloning Site: a short DNA sequence that contains many unique restriction enzyme sites and allows scientists to control the introduction of specific genes into the plasmid. Promoter: a DNA sequence that is typically located just before (“upstream” of) the coding se- quence of a gene. The promoter recruits RNA polymerase to the beginning of the gene sequence, where it can begin transcription.
Selectable Marker
Promoter
2.
Multiple cloning site
Plasmid Map
3.
Origin of Replication
Figure 2: Plasmid Features
4. Selectable marker: a gene that codes for resistance to a specific antibiotic (usually ampicil - lin, kanamycin or tetracycline). When using selective media, only cells containing the marker should grow into colonies, which allows researchers to easily identify cells that have been successfully transformed. TRANSFORMATION EFFICIENCY
final vol at recovery (mL) vol plated (mL)
Number of transformants µg of DNA
Number of transformants per µg
X
=
In practice, transformation is highly inefficient—only one in every 10,000 cells successfully incorporates the plasmid DNA. However, because many cells are used in a transformation experiment (about 1 x 10 9 cells), only a small number of cells must be transformed to achieve a positive outcome. If bacteria are transformed with a plasmid containing a selectable marker and plated on both selective and nonselective agar medium, we will observe very different
100 transformants 0.01 µg Specific example: X
100,000 (1 x 10 5 ) transformants per µg
1 mL 0.1 mL
=
Figure 3: Bacterial Transformation Efficiency Calculation
results. Nonselective agar plates will allow both transformed and untransformed bacteria to grow, forming a bacterial “lawn”. In contrast, on the selective agar plate, only transformed cells expressing the marker will grow, resulting in recovery of isolated colonies. Because each colony originates from a single transformed cell, we can calculate the transforma- tion efficiency, or the number of cells transformed per microgram (µg) of plasmid DNA (outlined in Figure 3). For example, if 10 nanograms (0.01 µg) of plasmid were used to transform one milliliter (mL) of cells, and plating 0.1 mL of this mixture (100 microliters, or 100 µL) gives rise to 100 colonies, then there must have been 1,000 bacteria in the one mL mixture. Dividing 1,000 transformants by 0.01 µg DNA means that the transformation efficiency would be 1 X 10 5 cells transformed per µg plasmid DNA. Transformation efficiency generally ranges from 1 x 10 5 to 1 x 10 8 cells transformed per µg plasmid.
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