Lab and Bioinformatics Sessions, Proteomics Experiments on Wheat Proteome and Cloning of Cancer-Expr

You have until 5.00pm on 12 th of January 2022 to complete the three questions on this paper, i.e., you must complete part A and both questions in part B. All questions are marked out of 100 and have equal weighting. Your final mark will be the average mark calculated from all three questions.

Your work must be uploaded via Turnitin to the assessment submission folder on Blackboard by 5.00pm on 12/01/22. You can check your Turnitin similarity score by submitting your draft assignment to the library submission area in ‘Assessment Support’.

This question relates to the lab and bioinformatics session and all information relating to this can be found in the folder ‘Lab & Bioinformatics Information’ on the Molecular Biology & Proteomics site on Blackboard.

1. The following questions all relate to the lab and bioinformatics sessions. If you were unable to attend in person, you should familiarise yourself with the lab protocols and work through the bioinformatics session using the detailed instructions in the MBP Module Booklet 20-21, which also contains comprehensive background information.

a. From the ‘control gel image’ on Blackboard state whether the recombinant plasmid samples A, B & C are based on pGEM-T or pGEM-T(Easy), stating your reasons. (10 marks)
b. From your database search, which chromosomes are the three NEIL genes from Mus musculus to be found? (10 marks)
c. How many nucleotides are in the mRNA sequence and the CDS sequence of NEIL1-isoform 1, NEIL2 and NEIL3? (20 marks)
d. State what the difference is between the mRNA sequence and the CDS sequence. (10 marks)
e. What is the paralog of the nei gene in E. coli and what are the functions of the two encoded proteins? State and difference in substrate preference. (20 marks)
f. Write a 500 word summary of the results of your homology and conserved domain searches. This should include results of the DNA and protein sequence comparisons and what the conserved domain information tells you. (30 marks)

Write two, 1000-word essays on the following two topics. Word count must be stated at the end of each essay. You are encouraged to include figures and diagrams but they must either
be original and your own, or the source cited, and the figure fully described in the text. You must refer to and cite appropriate literature used to inform your answer.

Word count: 1000 words. Content 10% over will not contribute to the determination of the awarded mark

1. The field of proteomics is evolving from cataloguing proteins under static conditions to making detailed comparative analyses. Defining proteins that change in abundance, form, location, or activity may indicate which proteins are involved in developmental changes or responses to alterations in environmental conditions. Such studies are facilitated by an increasing number of complementary technical options for performing quantitative proteomic comparisons. As with any developing field, however, rapid expansion in new techniques introduces concerns about choosing the appropriate approach.

As a research scientist you wish to understand plant responses to pathogen invasion. You have obtained an ancestral line of wheat with powdery mildew resistance.

Design a set of proteomics experiments to define pathogen responsive changes in the wheat leaf proteome. State your experimental aim clearly at the start of your essay. Your answer should consider the following points:

a. You should consider number of replicates (5 marks),
b. Experimental controls (5 marks),
c. Time points (5 marks),
d. Protein extraction method (15 marks),
e. You should decide upon a comparative analysis methodology and explain how the analysis is conducted including benefits of the approach and any technical limitations (50 marks),
f. Finally, in what format is the generated data and how would you interpret it? (20 marks)

2. You work in a cancer research laboratory and have discovered a protein that is expressed only in cancer cells and thus may be a good target for novel drug design. As a first step towards this goal, you need to express and purify substantial amounts of the protein in Escherichia coli in order to undertake further studies to design a specific small molecule inhibitor.

Your answer should consider the following points:
a. Given the structure of protein coding genes in mammalian cells, describe the process you would undertake to obtain the DNA coding sequence of the target protein. (30 marks)
b. Give an overview of the process of molecular cloning and the steps required to obtain a recombinant expression vector for the target protein. (30 marks)
c. Explain the different ways the pET vectors are optimised for inducible expression of recombinant proteins in E. coli. (30 marks)
d. Suggest one way of purifying the recombinant protein from the E. coli cell proteins. (10 marks)