Answer the questions provided in the document.
Assignment 3 – Generation of Recombinant DNAs with HIS tags - S21 Students must submit their completed work in Crowdmark. If this is not done by the deadline, your work will be marked as late. You must submit your work typewritten in either doc or pdf format, and submission of non-standard file formats or submissions of photos will receive a zero. Please ensure you give yourself enough time for proper submission and ensure that your answers are uploaded in the correct location, otherwise your work may not be graded. Answers that are ambiguous, unclear, or do not follow instructions will be penalized or receive no credit. Students are expected to work independently, and any work submitted must be your own. Students who post assignment content on group chats and third-party websites will be in violation of both the Senate Policy on Academic Honesty and/or the Canadian Copyright Act. Preamble Dr. Kovinich approaches you after you generate the primers to amplify a Gene with the following NCBI/FASTA Locus code (NM_001025248). He can’t remember what the gene is though so you’ll have to look it up. He wants you to amplify the CDS (same as ORF) sequence such that it can be cloned and then expressed in a pET15-b vector. He also remembers that there is more information on the NCBI site than needed for the CDS. He suggests that you accomplish this by the following workflow: 1. Engineering a NdeI or NcoI restriction enzyme recognition site (whichever is more appropriate) immediately before or at the start codon and a BamHI or XhoI recognition site immediately after the stop codon for the gene, using PCR. 2. Cutting both the PCR product and vector with your chosen restriction enzymes, and appropriately purifying the cut fragments that you wish to work with. You may assume that all restriction enzymes will work at 100% efficiency. 3. Using DNA ligase to join the cut insert and vector together, reforming a circular DNA with the PCR amplicon now incorporated into the vector within the MCS. 4. Remember! You will want to isolate the cloned expressed PCR product using a Ni Agarose column. Core Questions 1. (6 points) Provide the sequence of two primers that can be used to accomplish the task described above. For the sake of simplicity, each individual primer should be 25 nucleotides long. Provide these two sequences according to standard scientific conventions. Also indicate where the primers will bind (which strand and template complimentary sequence). 2. (4 points) A postdoctoral fellow in the lab looks over your shoulder at the cloning strategy that Dr. Kovinich has suggested and laughs at your plight. She tells you that the Tm of your primers is nearly the same when in fact they should be different as they are used to amplify different ends of the target template. Should you believe her? If yes explain why she is correct. If not, explain why she is wrong and why you are right. Provide your answer in 4 sentences max 3. (4 points) After cloning and transforming, you want to be able to isolate the plasmids from the transformant colonies to ensure that the PCR product was inserted into the plasmid and that it was in the right orientation. Describe how you might do this using a) Restriction enzymes b) PCR Be specific! Ie which RE and why (how do they prove insertion and orientation) and which primers (if any) and why. 4. (8 points) Before your ligation, you need to determine the proper amounts of DNA to use. You have been told to use 50 ng of pET 15b. The stock solution that you have is 0.75 mg/ml. Your amplified PCR product is determined to be 1 mg/ml. You MUST show your work. A simple answer will receive a zero grade. a) What volume of the vector should you use to get 50 ng? Hint – look at Part c below! [2] b) Knowing that you should have a 3:1 molar ratio of insert to vector, how much (volume) of the PCR product should you use to ensure these ratios? [4] c) Would you need to adjust the DNA concentrations in any way prior to the ligation? Clearly explain. [2] 5. You take your newly ligated plasmid and use it in a transformation reaction to put it into bacteria. You spread this transformation mixture out onto petri plates, along with a few other parallel reactions and control plates that you made as directed by Dr. Kovinich. These plates are listed below: Is there ampicillin on the plate? What was plated? Is there any growth? Plate A N Cells Transformed with Water (no plasmid) Y Plate B N Water Alone N Plate C Y Cells Transformed with Plasmid 100’s of colonies Plate D N Cells Transformed with Plasmid Lawn of bacteria Plate E Y Water Alone N a. (2 points) Which plate should in theory, exclusively contain bacteria that are transformed with your plasmid? Explain why in one sentence. b. (2 points) Which plate provides the best evidence that the bacterial cells you are using in your experiment are viable? Explain why in one sentence. c. (2 points) Which plate should provide the best evidence to determine if your experiment was contaminated by random bacteria in the lab environment? If you believe there isn’t a plate demonstrating this, then describe what you might plate to prove purity of the bacterial culture. Explain why in two sentences. d. (2 points) Which plate provides the best evidence that you likely did not get contamination with antibiotic-resistant bacteria from the lab environment throughout the course of your experiment? Explain why in one sentence. e. (4 points) Devise another control plate that provides evidence that untransformed cells do not already have the capability to resist ampicillin (because they shouldn’t). Indicate whether this plate should have ampicillin and what should be plated on it, in a similar fashion to the table above. Explain your reasoning in three grammatically correct sentences at most. f. (5 points) Later in the week, you repeated the experiment from the beginning but this time there were no colonies on Plate C. All the other plates had similar results compared to the first attempt. • Is this expected? Explain • Suggest and explain 2 reasons that might have occurred to lead to this result. • Your colleague suggests that the cells could not be transformed or died during the transformation procedure. What control could be used to prove that the cells could be transformed. Explain. Please explain concisely. Any errors in logical reasoning will receive no credit. 6. (7 points) You have isolated cells that appear to have the properly cloned PCR insert CDS. You then lyse the cells and run the lysate over a Ni-Agarose column. Upon running the eluate (solution eluted from the column) from the column over a an SDS-PAGE gel, you see that no protein has been isolated from your cells. Moreover, using UV spectroscopy at 280 nm (used to detect protein in solution), there appears to be no protein in the Ni-Ag eluate. Assuming that the recombinant plasmid is constructed properly, describe 3 reasons that could account for the lack of protein. How might you set up an experiment to test your hypotheses? BIOL2071 Assignment 4: Due August 10th 2021 The questions in this assignment are based on lectures and activities from this course. As such they should be answered based on the information provided in the course. Marks for each question are in brackets. 1. You are testing a newly synthesized hormonal analogue (called B23) for the ability to increase expression of the Biologase gene in humans. You decide to use qPCR to analyze expression in Red Blood Cells and in Liver cells. The cells are grown as pure cultures in the lab and are not sampled from living humans. You expose the cells (separately) to B23 and then take samples of the cells to lyse (at the times indicated in the graph) and then check Biologase expression. The data from the experiment is below. Time after exposure to B23 in hours Observed Ct Liver Cell Biologase Observed Ct RBC Biologase Observed Ct GAPDH 0 27.3 28.1 17.5 0 26.9 27.9 17.8 0 27.2 28.4 17.4 4 15.1 27.8 17.2 4 15.4 27.9 17.5 4 14.9 28.1 16.9 8 10.3 27.8 18.1 8 10.5 27.6 17.8 8 10.6 28 17.7 24 11.7 27.7 17.3 24 11.9 27.5 18.2 24 12 27.9 17.5 48 17.5 27.7 17.4 48 27.5 35.2 26.9 48 18.1 28.1 17.7 Answer the following based on this experiment and these results. a) Explain why GAPDH results were included in this table with reference to how and why it was used in the experiment. [2] b) Analyze the data and explain what the results tell you by directly referring to the results and how they support your analysis. Are there any anomalies? If so point them out and explain how they may have occurred. Explain this as you were explaining to a supervisor that you were trying to impress. [4] c) Is there a problem using pure cell cultures instead of testing B23 in situ? What is the value of testing in vitro? [2] d) Why do you think the timed samples went from 8 hours to the next one at 24 hours? Why might this be a (good or bad – pick one) idea? [2] 2. LAMP amplification is similar to PCR yet there are differences. Use your considerable literary researching skills to discuss why LAMP may be a preferred tool for COVID detection compared to normal or qPCR. Find reliable and citable sources to support your discussion. Make sure to make reference to the qualities of the respective techniques in your comparison. Also include a situation where qPCR would be preferred over LAMP and why. Use no more than one page maximum! [4]