Out of the Blue CRISPR

Name:_Saanvi Amara___________Period:____7____ Out of the Blue: CRISPR Lab Lab Grade:_____/14 ___Correct use of lab equipment​ ​ ​ ___Follows SOP (standard operating protocols) ___Follows sterile protocols​ ​ ​ ​ ___Correct lab technique ___Cleans up lab station​ ​ ​ ___Manages waste correctly ​ ___Follows safety protocols Part 1. Answer Pre-Laboratory Questions Use your notes on CRISPR gene editing along with the explanation of this lab (the lab sheet) to help you answer the following questions. 1.​ Explain how the components of the CRISPR system work together to edit the lacZ gene in this lab. Include Cas9 and sgRNA in your explanation. Underline these vocabulary words in your answer. The sgRNA has a guiding region which is complementary to a specific sequence in the lacZ gene. This allows the sgRNA to guide the Cas9 enzyme to the spot on the bacterial chromosome. After the sgRNA binds to the specific DNA sequence, and if the PAM sequence is present downstream, the Cas9 enzyme will cut both strands of DNA at that location. This double-strand break will activate the HDR repair system, which we will turn on using arabinose, so that the donor template DNA from the plasmid introduced can be used to repair the cut. The donor template we are introducing contains a stop codon so that when the cell repairs the cut using it, the lacZ gene is disrupted and will no longer produce the β-gal. We will know it was functional through the lack of blue colonies 2.​ Which type of repair mechanism is used in this lab? How does this repair mechanism work? The lab uses HDR, or homology directed repair, which is a precise DNA repair pathway. Once Cas9 makes the double-strand cut in the lacZ gene, the cell looks for the DNA template to use in repair. This template, provided through the plasmid, will be copied by the HDR machinery. We will activate the repair mechanism through arabinose, and with the template compiled, the stop codon will be inserted into the chromosome. Since the repair integrates this template, the lacZ gene is disrupted and can no longer produce β-gal. 3.​ What is the second type of repair mechanism (we did not use in this lab) that can be used with CRISPR editing? How does this repair mechanism work? The other type of repair mechanism is NHEJ, or nonhomologous end joining. This repairs a double-stranded break by reconnecting the two broken DNA strands, without using a template or inserting any new DNA. Because the preparation is quick and lacks structure, it often results in mutations such as insertions or deletions. These mutations can negatively impact or mutate the gene. It is often avoided to ensure there are no harmful mutations. 4.​ Draw a diagram and write an explanation that explains why the E. coli in this investigation are blue. Include the lacZ gene, β-gal, X-gal and IPTG. The E. coli in this investigation are blue because their lacZ gene is functioning normally and producing the β-gal enzyme. When IPTG is added to the plate, it serves as an inducer that turns on the lacZ gene, allowing the bacteria to make the β-gal. This enzyme then breaks down X-gal, present in the plate, into a blue product. This results in colonies with an active lacZ gene appear blue on the plates 5.​ What is the purpose of each of the two antibiotics (kanamycin and spectinomycin) used in this investigation (explain for each one)? Kanamycin is used to ensure that only E. coli that we began with grows on the petri dishes. The E. coli strand we start with, HB101-pBRKan, is naturally resistant to kanamycin (kan). This means that any bacteria potentially contaminating the dish won’t survive and ruin the experiment. Spectinomycin (SPT) is used to select for the bacteria which successfully took up the plasmid during transformation. Both plasmids being used carry a SPT-resistant gene, ensuring that only the bacteria that absorbed the plasmid and have the DNA template will survive and be used to quantify success. 6.​ Why will arabinose be used on the plates that you got your starting (non CRISPR edited) bacteria from? If arabinose is not on that plate, how will it affect the lab? Arabinose is used on the plates as it activates the HDR system in the E. coli strains. This bacteria already contains the Casi enzyme, so if it makes the cut without the HDR system turned on, the cells can’t repair damage and will die. The addition of arabinose ensures the HDR system is activated and the repair can truly happen. If arabinose was not on the plate, the HDR mechanism would remain inactive, meaning that when the Cas9 enzyme cuts the lacZ gene, the bacteria would be unable to repair the break. This would result in the cells dying instead of being edited, resulting in a failed experiment with no cell growth. 7.​ What is bacterial transformation used for? Why do scientists do it? Bacterial transformation is used to get bacteria to take in the foreign DNA, such as the plasmid we are inserting. In this lab, the plasmids carry the sgRNA needed for the CRISPR editing and HDR, so by transforming the cells we can ensure they have the genes they need to complete the lab. Bacterial transformation is done by flash heating the bacteria to make it competent, create holes plasmid can enter through, and then introducing them to a calcium chloride to neutralize the chemical charges and further aid in the intake of plasmids. Scientists do it to ensure that they can properly introduce new DNA templates to bacteria and maximize success of CRISPR experiments. 8.​ To transform bacteria they must be “competent”. What does this mean and how will we do it in this lab (two things)? Bacteria being competent means they have been treated and are able to take in plasmids. In order to do so, we will treat them with CaCl2 to neutralize the negative charges and loosen the membrane, and then we will heat shock the bacteria to create holes in the DNA that the plasmid can enter through. 9.​ In this lab, we are transforming bacteria with two plasmids (pLZDonorGuide, and pLZDonor). a.​ What is the point of inserting the pLZDonorGuide plasmid? What specifically is the Donor? The pLZDonorGuide plasmid is the plasmid containing the sgRNA gene and the donor template DNA. The sgRNA guides the Cas9 enzyme into the point of insertion so the Cas9 enzyme can cut the sequence out, however, without the donor template the HDR won’t be able to properly insert the stop codon effectively. The door is the donor template DNA carrier in the plasmid, containing a stop codon and DNA sequences that match the regions on both sides of the Cas9 cut site, which will be copied during HDR and edit the lacZ gene. b.​ What is the point of inserting the pLZDonor plasmid? Explain why we used this in our lab. The pLZDonor plasmid does not contain the sgRNA, allowing it to be used as a control in this experiment. Because it does not contain the sgRNA to guide the Cas9 to the proper spot in the lacZ gene, the gene won’t be cut so HDR can’t be triggered and genetic modification won’t occur. pLZDonor shows what happens when bacteria only receive the donor DNA without CRISPR targeting, allowing us to compare the Cas9 effects on genetic engineering. Since it serves as a control group, we’ll be able to ensure that our experiment occurred properly as CRISPR editing can only happen with the sgRNA present. 10.​In the table below, predict what you would expect to grow on each of the plates. HYPOTHESIS Plasmid Plate Bacterial Growth (yes/no) Colony Color (+) pLZ Donor Guide IX/SPT yes white (-) pLZ Donor Only IX/SPT yes blue 11.​ If no plasmid were added and the bacteria plated on the same IX/SPT (IPTG, X-gal, spectinomycin plates), what would you expect to happen? If no plasmid were added and the bacteria was plated on the same IX/SPT plates, the bacteria would not grow. This is because the plates contain SPT and the only way for the bacteria to continue growing in the presence of SPT is if they contain the plasmid, which carries the SPT resistant gene. I would expect there to be no colonies under these circumstances. Since the SPT will kill all the bacteria, as they don’t contain the plasmid introduced SPT resistance, there will be nothing to grow. 12.​If the bacteria does not have a functional lacZ gene, what color would you expect the colonies to be? If the bacteria don’t have a functional lacZ gene, I would expect the colonies to be white. This is because when functioning normally, the lacZ gene makes β‑gal, which breaks down the X-gal present in the tray and turns the colonies white. However, if the lacZ gene is edited and no longer functions, β‑gal won’t be produced and the tray will remain white. Part 2. Answer the Post Lab Questions 1.​ Complete the following data table for your two plates. RESULTS Plate # Blue Colonies # White Colonies Total # Colonies % of White Colonies (#White/#Total) Comparison with Hypothesis + - 2.​ Explain how colony color can be used as evidence of the state of the lacZ gene in the bacteria. 3.​ Did your plates show evidence that the CRISPR system edited the lacZ gene? Explain. 4.​ What was the efficiency of the CRISPR system in editing the lacZ gene in this lab? Explain. To calculate this follow the directions on your lab sheet. Explain. 5.​ If you have any unexpected results, list them here and provide a hypothesis for how/why they occurred. 6.​ What is another method (other than the blue white screen) that we could use to verify that the gene was edited in the bacteria? Explain how it works.