Genetics review

An undergraduate student, Aimin, has joined the research lab where you are the senior undergraduate researcher. They have proposed a UROP project (Undergraduate Research Opportunities Program) in which they will explore the role of a histidine (His5) that lies in the active site of an enzyme, finalase. Finalase enables organisms to combat the effects of stress. Finalase catalyzes the breakdown a stress hormone commonly referred to as “AHHHH”. His5 is completely conserved among the finalases from different species. Aimin’s UROP proposal intends to test this theory by designing, making and testing a site-directed mutant of the Arabidopsis thaliana finalase (i.e. where His5 is replaced by another amino acid: the rest of the protein remains the same). A. thaliana is a plant species regularly used to do genetics research. You are working with the student and guiding them in their UROP research. 4A. (4 points) First, research the species Arabidopsis thaliana. Describe at least two characteristics of this plant species (or the knowledge we have about it) that make it a good species to use for conducting genetic modification experiments. When listing the two characteristics, make sure to describe WHY each of these characteristics makes A. thaliana a good study species for genetics research. Cite your sources using proper APA 7th edition citation format. (2-5 sentences) 4B. (6 points) Aimin makes two suggestions of different amino acids that they could replace His5 with to test its catalytic role in finalase: Lys and Leu. You tell Aimin these are excellent choices. a. What do you think is the main chemical/physical property of histidine that Aimin is testing through each of these choices of amino acid substitutions? (2 sentences) b. Assume the property being tested in each case is the only characteristic relevant to finalase’s function. Would finalase be functional or non-functional based on the similarities/differences between histidine and each amino acid? Why? (2-3 sentences) Remember to use the resources provided with the exam. Example answers: If His5 is replaced with Tyr, Aimin is testing how important a ring structure in the side-chain is in the function of His5 in finalase. If this property defines the role of His5 in catalysis, then the mutant finalase would be functional as both His and Tyr have ring structures. If His5 is replaced with Gly, Aimin is testing how important amino acid size/possessing a side-chain is in the function of His5 in finalse catalysis. If this is the key property that His5 brings to finalase activity, then the mutant finalase would be non-functional as Gly is so much smaller/has no side-chain compared to His. 4C. (6 points) Aimin’s final choice is to replace His5 with Lys. The beginning and end of the wild-type finalase gene are contained within the following 5’ to 3’ nucleotide sequence in the A. thaliana genome. You can use highlighting, arrows, or some other diagram to provide your answers to this question, but each person must provide their own image if uploading something separate. a. Indicate the start of transcription and the end of transcription (Note: the sequence may contain DNA upstream of the start and downstream of the end). b. Determine the amino acid sequence for the beginning and end of the finalase enzyme before and after the 100 amino acid codons). c. Indicate where the codon for His5 is in the sequences. d. State the codon sequence you would use to replace His5 by Lys. 5’-tataatcgcgatgactaatcctcatcgaagtggcgtt……[100 amino acid codons]…. …catcggtatgtttaagcaatgcg-3’ 4D. (5 points) Aimin is trying to decide whether they should use CRISPR-Cas9 as the method to introduce the mutation into the plant or agrobacterium. (3-5 sentences total) a. Which method would you recommend that Aimin use and why? Be specific. b. For whichever method you chose, how many plasmids will Aimin need to design? Explain the role of every plasmid that is needed in the chosen method. 4E. (6 points) Aimin believes they have correctly introduced the mutated finalase gene into A. thaliana. Help Aimin design two primers of 8 bases each so that PCR and subsequence sequencing can be used to check if the mutation has successfully been introduced. Make sure to indicate which ends of each primer are 5’ and 3’. 4F. (3 points) Aimin finds that the mutation has been successfully introduced into the A. thaliana genome based on their sequencing results. However, they want to confirm that the His5 that they mutated was actually necessary for finalase to function. Briefly describe an experiment that Aimin could do to determine the impact of their mutation. Remember to consider the elements that make for a good experiment. (3-5 sentences)