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)