EDVOTEK® Workshops - 2024 NSTA Denver

EDVOTEK Workshops offered at the 2024 NSTA National Conference in Denver. Thursday & Friday March 21 & 22. 1. Forensic Escape Room: Design Your Own Biotech Adventure 2. Code Breakers: Using CRISPR to Rewrite Genetics 3. The Sweet Laboratory: Exploring Food Science with Biotechnology 4. Put the M into STEM: Quantitative Techniques for Biotechnology 5. Heavy Metal: Investigating the Effects of Environmental Toxins on C. elegans 6. Introducing Your Students to CRISPR with Sickle Cell Gene Editing 7. Lion Family Reunion: Conservation Biology Genetics

EDVOTEK® WORKSHOPS • 2024 NSTA DENVER

TABLE OF CONTENTS

WORKSHOP

PAGE

01 Forensic Escape Room: Design Your Own Biotech Adventure

02 Code Breakers: Using CRISPR to Rewrite Genetics

15 29 41 53 65 77

03 The Sweet Laboratory: Exploring Food Science with Biotechnology 04 Put the M into STEM: Quantitative Techniques for Biotechnology 05 Heavy Metal: Investigating the Effects of Environmental Toxins on C. elegans 06 Introducing Your Students to CRISPR with Sickle Cell Gene Editing

07 Lion Family Reunion: Conservation Biology Genetics

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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 Laura Smith has been arrested for being the main suspect in the murder of her husband, Jeremy Smith. She has been questioned multiple times and continues to plead innocent to the crime. Laura’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 blood 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 Laura’s innocence? The following information may help you with your forensic assessment of the crime. On Saturday night, police dispatch received a distressed phone call from a woman in her mid-30’s, claiming to have found her husband dead in their kitchen upon returning home. Dispatch sent officers to the scene shortly after. Clay, the local police sheriff, was called down to the murder scene at the small suburban condo in Charleston, South Carolina. When he arrived at the condo, Clay found a deceased man in the kitchen. There was a major head laceration on the deceased man. Upon initial inspection of the scene the following primary evidence was collected: glass shards (from a broken bottle of red wine) 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 wine glass with women’s lipstick on it. Clay and his crime scene investigators collected the evidence from the scene and the coroner was able to identify the body as Jeremy Smith. Jeremy Smith was a 32 year old man, who was married to 33 year old Laura Smith. Laura was the dis- tressed woman from the police dispatch phone call. When questioned about her whereabouts during the crime, she claimed to have gone to an urgent care to take care of a deep wound she obtained. Laura insists that she accidentally cut herself while cooking dinner with Jeremy. Clay and his partner Amy, questioned the couples’ neighbors. Most of the neighbors complained about their constant arguments. Their fights often disrupted the peaceful neighborhood. However, neighbors also noted that Jeremy was also constantly fighting with the HOA president, Sarah Ann. She is known for being a stickler on the HOA rules and ever since the Smiths moved in, there have only been fights between Jeremy and Sarah. The meeting notes from the HOA meetings prove that the two have a history of being verbally aggressive towards each other.

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Clay and Amy have determined that Laura and Sarah Ann are the main suspects of this crime. The head laceration was determined to be what caused Jeremy’s death. The glass shards in Jeremy’s head wound matched the broken glass from the wine bottle found at the crime scene. According to eyewitness re- ports, Laura and Sarah Ann were seen at the scene of the crime 1 hour prior to the crime. Alcohol was detected in both Sarah Ann and Laura’s blood samples. It was confirmed by the local urgent care that Laura was treated for a knife wound.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

1155

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Introduction Unleash the power of gene editing with your students using REAL CRISPR-Cas technology to knock out colorful genes in E. coli. Identify successful knockout based on the cell color. Experi- ment by switching RNA templates and analyzing results, letting your students prove the specific - ity of CRISPR! Background Information CRISPR is a game-changing gene-editing tool that is catalyzing a wave of innovations across fields as diverse as biotechnology, medicine, agriculture, and energy. This system evolved in bacteria at the beginning of evolutionary history as a defense against viral attacks and was discovered by humans in the late 1980s. However, it would take scientists another 20 years to harness CRISPR as a precise and versatile gene editor. Today, CRISPR’s unmatched ability to rewrite the code of life has captured the imagination of re- searchers and the public alike. CRISPR technologies are used to cure diseases like cancer and sickle cell anemia, develop antibiotics, study complex genetic traits, understand evolutionary history, create drought-resistant crops, and develop new materials like bioplastics and biofuels. As CRISPR continues to advance its transformative potential will be limited only by our imagination and by the regulations we create. WHERE DOES THE NAME CRISPR COME FROM? CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats describes uniquely patterned regions of bacterial DNA. An enzyme tightly linked to these CRISPR region was identified by scientists and called Cas for “CRISPR-associated proteins”. In bacteria and archaea, Cas enzymes cut and destroy foreign DNA, guided by the nucleotide sequences stored in CRISPR regions. Cas enzymes

are central to new DNA editing technologies but the CRISPR region itself is not. Never the less the CRISPR name has be- come synonymous with these technologies. EXPLORING THE CRISPR-CAS SYSTEM At the heart of CRISPR gene editing are two molecules: (1) an enzyme called Cas and (2) a

Figure 1: Bacterial CRISPR Region.

piece of RNA known as guide RNA (gRNA). These molecules collaborate to form a complex capable of identifying and cutting a specific DNA sequence within a genome, known as the target DNA. Following a cut, a cell’s intrinsic repair mechanisms mend the targeted DNA often with the help of additional DNA instructions, called template DNAs, that the experimenter provides. This repair process leads to changes in the DNA sequence that can induce a loss of function mutation, correct a mutation, or introduce a new trait in an organism. Cas proteins are endonucleases. These are enzymes that cut DNA by breaking the phosphodiester bonds between nucleotides. Some endonuclease cut DNA nonspecifically, but most cleave at specific nucleotide sequences called target sites. Cas enzymes fall into this latter category. They target specific sites within a DNA molecule and create a double-strand break. However, unlike most endonucle- ases (like restriction enzymes), which have a target DNA sequence built into their structure, CRISPR outsources its specificity to interchangeable molecules of guide RNA. This means that Cas enzymes can be programmed to cut any DNA region by creating guide RNA molecules that are complementary to the target’s sequence.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Guide RNAs (gRNA), sometimes called single guide RNA (sgRNA),are the second key component for CRISPR gene editing. Serving as ‘molecular guides’, they direct the Cas enzymes to a specific target site within a genome. Guide RNA molecules consist of two functional parts. The first is a scaffolding sequence where the Cas enzyme binds. The second is an approximately 20-nucleotide sequence which determines where the DNA will be cut. Through base-pairing interactions, this nucleotide

sequence aligns with the target DNA and brings the Cas enzyme to the intended cut site. To ensure proper interactions between the gRNA - CAS complex and the target DNA, there’s an ad- ditional requirement: a nearby DNA sequence known as a protospacer adjacent motif, or PAM, sequence in the organism’s DNA. This is a sequence of two to six nucleotides found immediately downstream of the DNA sequence targeted

Figure 2: Target DNA and PAM site.

Cas9

gRNA

by the gRNA. PAM sequences allow the CAS enzyme to properly bind to, scan, and cut DNA. However, they also limit what DNA regions can be targeted. For example, the popular CRISPR enzyme Cas9 requires a 5′-NGG-3′ PAM sequence (where “N” can be any nucleotide base). This means that for a researcher using Cas9 the genomic locations that can be targeted are restricted to those containing a downstream 5′-NGG-3′ sequence. Luckily, there are a range of CAS enzymes each with its own unique PAM sequences. Multiple CAS nucleases have been identi- fied. Cas9 is a prominent example. Originally discovered in Streptococcus pyogenes, Cas9 quickly gained popularity in CRISPR gene ed- iting due to its high efficiency and accuracy. Other commonly used CRISPR endonucle- ases include Cas3, Cas12, Cas13, casino, SuperFI-Cas9, and Cs7-100.

Binding to target

Double-stranded cut

Figure 3: CRISPR targeting and digestion of DNA.

The target DNA represents the final critical component for the CRISPR experiment. This is

the specific sequence of DNA that researchers aim to modify. During a CRISPR experiment, the CAS- gRNA complex scans an organism’s DNA by unwinding short segments of DNA and comparing these sequences to the gRNA’s 20 bp binding sequence. When the complex reaches a region of DNA that is complimentary, it attaches. Once attached the Cas enzyme cuts the DNA. THE FORK IN THE ROAD: HOMOLOGOUS DIRECT REPAIR AND NON-HOMOLOGOUS END JOINING Once a CAS enzyme has cut through the double stranded DNA, the cell fixes the damaged nucleic acid chain using its intrinsic DNA repair pathways. Researchers design their CRISPR experiments so that the cell repairs the DNA in one of two ways: using non-homologous end joining (NHEJ) or homologous direct repair (HDR). These two repair mechanisms produce different results, allowing researchers to change the target DNA in distinct ways.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

NHEJ s a simple but error-prone process that occurs naturally in cells and is used in the lab for knock- out experiments. These are experiments that study the function of a gene by inactivating it with a loss of function mutation and then observing the results. When Cas cuts through the DNA backbone, the cell’s most direct means of repair is to join the two broken ends of DNA back together. However, this process usually results in small deletions or insertions at the cut site which cause frameshift mutations that disrupt the function of the targeted region. Unlike classical knockout methods, which required screening thousands of random mutations, CRISPR knockout experiments allow research- ers to design guide RNA that automatically targets their gene of interest, streamlining the process by eliminating one of the most laborious and time-intensive steps. HDR is also a naturally occurring process but more precise, controlled, and complex than NHEJ. It involves mending the double-stranded break using a donor or template DNA as a reference. To work properly, this template DNA must contain regions that match the target DNA sequences on both sides of the break. In cells, this is often the sister chromosome, which shares the same sequence. In CRISPR experiments, researchers develop a repair template that matches the genomic DNA around the cut site. However, in between these homologous beginning and ending regions, scientists can introduce specific mutations or new genes. As the cell repairs itself, these insertions are transcribed from the template to the target DNA allowing for specific and user-designed changes to an organ - ism’s genome.

Figure 4 - Repairing DNA Using HDR

CRISPR-Cas targeting DNA

Corrected DNA sequence

Homologous DNA sequences

Homology Directed Repair (HDR)

Repaired DNA

Figure 4: Repairing DNA Using HDR

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Summary CELLS:

BactoBeads E. coli pChromoPurple: The host bacteria have been genetically engineered to produce the purple protein. First, the E.coli genome has been modified to contain the gene for T7 polymerase under the control of the lac operon. Next, the modified bacteria are transformed with a plasmid containing the Purple Chromoprotein under the control of the T7 promoter. When IPTG is added, derepressing the lac operon, the cell can synthesize T7 polymerase. The poly- merase binds to the plasmid and turns on production of the purple protein. BactoBeads E. coli HB101: The host bacteria’s genome has been genetically engineered to express the beta-galacto- sidase protein under the control of the lac operon. When IPTG is added, derepressing the lac operon, the cell produces the beta-gal protein. This enzyme cleaves X-gal into bioprod- ucts that cause the colonies to change from white to blue. PLASMIDS: pCas-purple This plasmid contains the CAS gene, the chloramphenicol resistance gene, and a sgRNA that targets the purple Chromoprotein mRNA. pCas-Blue This plasmid contains the CAS gene, the chloramphenicol resistance gene, and a sgRNA that targets the beta-gal mRNA.

pCas-sgRNA

WHAT WILL HAPPEN? Write your hypotheses below. Set 1: E coli Chomopurple + pCas-purple

Target gene DNA

pCas-sgRNA

Set 2: E coli Chomopurple + pCas-blue:

Set 3: E coli HB101 + pCas-blue:

Set 4: E coli HB101 + pCas-purple:

Bacterial Colonies

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Designing gRNA to Target LacZ

1. IDENTIFY and HIGHLIGHT five PAM sites in the target sequence. For this experiment, assume that you are using a Cas9 enzyme which uses an 5’-NGG-3’ PAM site. In this notation, the “N” can be any nucleotide. This means that the Cas9 will only bind to sequences immediately upstream (in the 5’ direction) of an AGG, TGG, CGG, or GGG sequence. NOTE: Since Cas9 can bind to either of the complementary DNA strands, remember to examine both for PAM sequences. 2. IDENTIFY and UNDERLINE the binding regions upstream of your highlighted PAM sites. In CRISPR, the guide RNA recognizes and binds to 20 nucleotides on the DNA strand opposite from the NGG PAM site. Binding always occurs upstream (in the 5’ direction) of the PAM sites. 3. Using base pairing rules, CREATE a guide sequence for the gRNA. This sequence should bind to the underlined region identified in step 2. For complimentary binding, Adenine (A) pairs with Thymine (T) and Cytosine (C) pairs with Guanine (G). Remember that for the bottom strand, a 5’ to 3’ direction means recording the sequence from right to left. 4. USING steps 1-3, DESIGN five candidate gRNAs for the LacZ Gene Sequence (0-200 bp). RECORD your answers in Table 2.

EXAMPLE SEQUENCE: 5’-TTCACTGCGTTCAGCAAAAAGTGAATTCTTGGTTACTGC 3’-AAGTGACGCAAGTCGTTTTTCACTTAAGAACCAATGACG

LacZ Gene Sequence (0-200 bp) 5’- ATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTG 3’- TACTGGTACTAATGCCTAAGTGACCGGCAGCAAAATGTTGCAGCACTGAC GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT CCTTTTGGGACCGCAATGGGTTGAATTAGCGGAACGTCGTGTAGGGGGAA TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA AGCGGTCGACCGCATTATCGCTTCTCCGGGCGTGGCTAGCGGGAAGGGAA CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACC - 3’ GTCAACGCGTCGGACTTACCGCTTACCGCGAAACGGACCAAAGGCCGTGG - 5’

Guide Sequence TABLE 2

PAM Sequence

gRNA Name Example gRNA 1 gRNA 2 gRNA 3 gRNA 4

TTCAGCAAAAAGTGAATTCT

TGG

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Procedure: Transformation

150 µL ice-cold CaCl 2

10 µL Plasmid (See table 2)

MIX

42°C

37°C

150 µL SOC to each tube.

Gently flick to mix.

NOTE: Keep tubes on ice as much as possible during this module.

1. COLLECT your four tubes of competent cells. LABEL the two purple bacteria tubes "1" and "2" and the two blue bacteria tubes "3" and "4" (see Table 1). ENSURE that the cells are kept on ice at all times. 2. Using a new pipette tip for each tube, ADD 150 µL ice cold CaCl 2 solution to each tube and MIX by gently pipetting up and down several times. 3. Using a new pipette tip for each tube, ADD 10 µL of the appropriate plasmids to each tube (see Table 1). MIX by gently pipetting up and down seveal times. 4. INCUBATE tubes on ice for 30 minutes. 5. Quickly PLACE the transformation tubes in a 42 °C water bath for exactly 45 seconds. 6. Immediately RETURN the tubes to the ice bucket and INCUBATE for 2 minutes. 7. Using a new pipette tip for each tube, ADD 150 µL of SOC solution to each tube. MIX by gently flicking each tube. 8. PLACE tubes in a 37 °C water bath and INCUBATE for 60 minutes to allow for recovery. continued

TABLE 1

Experiment 1 2 3 4

Bacteria E. coli pt Purple E. coli pt Purple

Plasmid pCas-Purple pCas-blue pCas-Purple pCas-blue

Plate Amp/IPTG/Chloramphenicol Amp/IPTG/Chloramphenicol Xgal/IPTG/Chloramphenicol Xgal/IPTG/Chloramphenicol

E. coli Blue E. coli Blue

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Procedure: Transformation

150 µL

REMOVE all but 150 µL

MIX

5000 rpm

VISUALIZE and RECORD

ROTATE & SPREAD

SPREAD the cells

COVER

37 °C OVERNIGHT

9. While cells are recovering, COLLECT and LABEL the bottom of four agar plates as indi- cated below. NOTE: Keep writing small and along the edge so you can easily see your colonies at the end.

• Purple pCas-Purple or 1 (Amp/IPTG/Chloramphenicol plate) • Purple pCas-blue or 2 (Amp/IPTG/Chloramphenicol plate) • Blue pCas-Purple or 3 (Xgal/IPTG/Chloramphenicol plate) • Blue pCas-blue or 4 (Xgal/IPTG/Chloramphenicol plate) 10. Following recovery, SPIN the tubes at 5000 rpm for 1 minute. 11. PLATE your four transformed cell lines by performing the fol- lowing five steps one tube at a time. Use a new sterile pipet tip and loop for each of tube/plate. (a) REMOVE all but 150 μL of the media*. Work carefully during this step to making sure the cell pellet stays in place.

*STEP 11 NOTE: Estimate 150 μL using the 0.1 mL volume marker on the tube. If necessary, overestimate and leave slightly more than 150 μL during steps 11a and 11b rather than underestimate.

(b) Gently RESUSPEND the pellet in the remaining media by pipetting up and down several times until no clumps are visible. (c) COLLECT 150 μL of the resuspension and PIPET into the center of the appropriate agar plate. (d) Use a loop to SPREAD the cells evenly and thoroughly over the entire surface. (e) TURN the plate 90° and thoroughly SPREAD again. 12. COVER the plates with lids and INCUBATE at room temperature for 5 minutes to allow the liquid to be fully absorbed. 13. STACK the plates on top of one another and TAPE them together. INVERT plates (agar side on top) and PLACE in a 37°C bacterial incubation oven. INCUBATE overnight (24 hours). If you do not have an incubator, colonies will form at room temperature in ap- proximately 48 hours. 14. VISUALIZE the transformation and control plates and RECORD the number of colonies on the plate and the color(s) of the colonies. If the colors are faint, the plates can be left in the refrigerator (4°C) for 24-48 hours to for further color development.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Transformation Troubleshooting

CRISPR TROUBLESHOOTING GUIDE

PROBLEM:

CAUSE:

ANSWER:

Incubation time too short

Continue to incubate source plate at 37ºC for a total of 18-22 hours.

Poor cell growth on source plate

Incorrect incubation temperature

Use a thermometer to check incubator temperature. Adjust temp. to 37°C if necessary.

Ensure the correct concentration of antibiotic was added to plates - Make sure ReadyPour is cooled to 60°C before adding antibiotic. Make sure ReadyPour is cooled to 60°C before adding antibiotic.

Incorrect concentration of antibiotics in plates Antibiotic is degraded

Satellite colonies seen on experiment plate

Incubate the plates overnight at 37ºC (18-22 hours).

Plates were incubated too long

Plates containing transformants were inverted too soon

Allow cells to fully absorb into the medium before inverting plates.

Colonies appeared smeary on experiment plate

After pouring plates, allow them dry overnight at room temp. Alternatively, warm plates at 37°C for 30 min. before plating cells

Experimental plates too moist

Ensure plasmid DNA was added to tubes.

Plasmid DNA not added to transformation mix

Make sure that pipets are used properly and are properly calibrated.

Incorrect host cells used for experiment

Confirm that correct bacterial strain was used for transformation

No colonies seen on experiment plates

Cells were not properly heat shocked

Ensure that temp. was 42ºC & heat shock step took place for exactly 45 sec.

Incorrect antibiotics

Be certain that the correct antibiotic was used.

Completely resuspend the cells in the CaCl 2 , leaving no cell clumps (vortex or pipet up and down to fully resuspend cells). Cell suspension should be cloudy. Fully but gently resuspend cells following centrifugation by slowly pipetting up and down. Ensure CaCl 2 and CCS are cold. Swipe through a dense section of the bacterial culture and use a full match sized loop. Incubate for full times. Keep on ice. Important that source cells grow no longer than 20 hrs. Refrigerate plates after 20 hrs if necessary. Do not use source plates that have been incubated longer than 24 hours (refrigerated or not).

Cells not well resuspended in CaCl 2

Too many injured or non-competent cells.

Not enough cells used for experiment

Source plates were incubated for more than 20 hours

Experimental plates too old

Prepare plate and use shortly after preparation

Completely resuspend the cells in the CaCl 2 , leaving no cell clumps (vortex or pipet up and down to fully resuspend cells). Cell suspension should be cloudy. Pre-chill CaCl 2 before adding cells to the CaCl 2 Extend incubation of celll suspension on ice 10-15 min. (should not exceed 30 min. total). This increases the transformation efficiency. Ensure that correct volume of plasmid was added to the transformation tube. If using micropipets, make sure students practice using pipets. Ensure that temperature was 42ºC and that heat shock step took place for no more than 45 seconds.

Low transformation efficiency (only a few colonies seen on experiment plates)

Cells not well resuspended in CaCl 2

CaCl 2 solution not cold enough

Cell solution not cold enough

Too much or too little plasmid DNA added to cell suspension

Cells were not properly heat shocked

Antibiotics were degraded prior to pouring plates

Make sure ReadyPour is cooled to 60°C before adding antibiotic.

Incorrect concentration of antibiotics

Ensure that the correct concentration of antibiotic was used in plates.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Results and Analysis