Coursework Reassessment

Coursework Reassessment Coursework

(All Tasks must be performed INDEPENDENTLY)

Before attempting the questions, please review the learning materials on Blackboard. If you have any queries regarding this assessment please Email the module leader. You should not consult any other student who is retaking this coursework piece. Perform the tasks outlined below and submit them as a single Word document containing your results (copied text / images or screen prints) via Turnitin. Note – be clear, concise and selective about the data you show.

Task 1

1. You have been given a plasmid from a colleague’s project containing the CYP3A4 gene. Unfortunately, the paperwork containing the map of the plasmid has not been included in the information supplied to you. You will need to map this plasmid before you can continue with the project. You have set up a series of restriction digests. You obtained the following results using agarose gel electrophoresis (Table 1.1).

Table 1.1 Restriction digest enzymes and fragment sizes

Lane	Digest	Size of fragments in bp
1	Pst I and Cla I	3600, 3200
2	Cla I and EcoRI	6000, 800
3	EcoRI and Pst I	2800, 4000
4	EcoRI and Xba I	1200, 5600
5	Xba I and Cla I	4800, 2000
6	Cla I	6800
7	Pst I	6800
8	EcoRI	6800

1a) What is the approximate size of the plasmid, how did you arrive at this conclusion?

1b) Construct a plasmid map of the plasmid you have been supplied (you must show all steps not just the final map). Represent your answer as a circular plasmid with the restriction sites indicated by their corresponding letters. You must also indicate on your plasmid map the distances between each of the restriction sites. Assume the Cla I I site is at the 12 O’clock position on the plasmid.

Use the abbreviations: C= Cla I , E= EcoRI, P = Pst I and X= Xba I

Task 2

The section between the Cla I site and the Pst I site contains the clockwise 3200 bp CYP3A4 gene. Below is the sequence between the Cla I and Pst I sites of the plasmid.

~~~ denotes the 1000s of bps of DNA between the Cla I site and the Pst I site which is important but not required for this section of the exercise. Assume that codon frame is retained between the ~ ~ symbols. The Frame one codon frame has been indicted.

2a) Write out the complementary DNA sequence.

2b) Translate all 6 reading frames, are there any possible protein encoding regions. Indicate possible start and stop codons.

Task 3

You have been charged with the task of cloning the CYP3A4 gene (in the correct orientation using information from Task 1 & 2) into the pFastBacDual vector, so you can utilise the p10 promoter. This cannot be carried out via a direct restriction digest and ligation so you must devise a cloning strategy by designing a suitable pair of primers (containing new restriction digest sites) which you will use to amplify the CYP3A4 gene with and then clone in to the vector. (Hint you should use the MCS of the vector to aide your decision whilst making the primers).

Figure 1.1 shows a vector map of pFastBac Dual and the multiple cloning site downstream from the two types of promotors.

3a) Using the sequence shown in Task 2, you should use the MCS of the p10 promoter to guide the design of a 5’ primer and a 3’ primer. It is for you to decide what restriction site you build into your two primers and the length of the primer you design. If you have identified a start site and end site in the sequence in Task 2 you should endeavour to retain them in the respective primers. Be mindful of how the primers would need to be written down so they are made correctly when being synthesized.

Figure 1.1 Vector map of pFastBac Dual including a more detailed view of the MCS.

For this task you should design a 5’ primer and a 3’ primer. You should indicate any import features that you have built into your primer or that you have decided to keep and indicate the annealing temperature you will use in your PCR reaction. Think about how you will order the primers so that the primer binds to the correct portion of the sequence.

3b) Once you have obtained your primers you should describe using bullet points how you will extract the CYP3A4 gene from the original vector and how you will transfer it to the pFastbac^Tm Dual vector. Marks will be given for the clarity of your methodology.

3c) Once you have cloned the CYP3A4 gene into pFastBac1. What restriction digests would you carry out to determine that the ligation has worked (you should choose at least three to carry out your diagnostic digest)? Indicate what fragments you would obtain from the digests (this need only be approximate according to the information you have). Would this digest tell you whether the gene is in the correct orientation, if so why? How would you ensure that you cloned the correct fragment (the one containing the gene) from the vector?

Submission details

You should submit your work electronically via the turnitin link “PHAR5330 Reassessment Coursework” in the “Reassessment coursework 2020” folder within the “Assessment and feedback” folder on the PHAR5330 blackboard shell by the stated deadline.

Your submission should include your p number in footer of your word file and should also contain your p number in the name of the file itself but should not contain your name as this submission will be marked anonymously. You do not need to submit a printed copy of your work.

The deadline for submission of the Coursework reassessment is 4:00 pm on the 2^nd of September 2020.

Assessment and feedback

The assessment criteria for the mini project are:

Criterion	Descriptor	Marks available
Technical competence	Ability to successfully perform the tasks using an appropriate methodology	30
Appropriate evidence	Relevance of supporting evidence	20
Response to questions / Critical Analysis	Appropriate data extraction, accurate answers to the questions, correct interpretation and evaluation of the data set	50

Coursework reassessment tasks will be marking with reference to the marking scheme for postgraduate taught course (final page).

Feedback for the assessment will be posted on the blackboard shell via grademark within 20 working days of the submission date.

Postgraduate Taught Course Marking Descriptors

Mark Range	Criteria
90-100% Distinction	Demonstrates an exceptional ability and insight, indicating the highest level of technical competence The work has the potential to influence the forefront of the subject, and may be of publishable/exhibitable quality. Relevant generic skills are demonstrated at the highest possible standard.
80-89% Distinction	Demonstrates an outstanding ability and insight based on authoritative subject knowledge and a very high level of technical competence. The work is considered to be close to the forefront of the subject, and may be close to publishable/exhibitable quality. Relevant generic skills are demonstrated at a very high level.
70-79% Distinction	Demonstrates an authoritative, current subject knowledge and a high level of technical competence. The work is accurate and extensively supported by appropriate evidence. It may show some originality. Clear evidence of capacity to reflect critically and deal with ambiguity in the data. Relevant generic skills are demonstrated at a high level.
60-69% Merit	The work is well developed and coherent; may show some originality. Clear evidence of capacity to reflect critically. Relevant generic skills are demonstrated at a good level.
50-59% Pass	Demonstrates a sound, current subject knowledge. No significant errors in the application of concepts or appropriate techniques. May contain some minor flaws. Demonstrates satisfactory subject knowledge. Some evident weaknesses; possibly shown by conceptual gaps, or limited use of appropriate techniques. The work is generally sound but tends toward the factual or derivative. Limited evidence of capacity to reflect critically. Relevant generic skills are generally at a satisfactory level.
40-49% Fail	Demonstrates limited core subject knowledge. Some important weaknesses; possibly shown by factual errors, conceptual gaps, or limited use of appropriate techniques. The work lacks sound development. Little evidence of capacity to reflect critically. The quality of the relevant generic skills do not meet the requirements of the task. Demonstrates inadequate subject knowledge.
30-39% Fail	The work lacks coherence and evidence of capacity to reflect critically. The quality of the relevant generic skills do not meet the requirements of the task. Demonstrates seriously inadequate knowledge of the subject.
20-29% Fail	The work contains minimal evidence of awareness of relevant issues or theory. The quality of the relevant generic skills do not meet the requirements of the task.
10-19% Fail	The work is almost entirely lacking in evidence of knowledge of the subject. No evidence of awareness of relevant issues or theory. The quality of the relevant generic skills do not meet the requirements of the task.
0-9% Fail	The work presents information that is irrelevant and unconnected to the task. No evident awareness of appropriate principles, theories, evidence and techniques.