This assessment evaluates your understanding of the learning objectives and activities covered from weeks 1 to 5, inclusive, in the unit. This includes pre-tutorial materials such as weekly lecture notes, videos, and other relevant resources provided with the unit content, as well as topics covered during scheduled classes. The assessment will consist of various question formats, such as short answer questions, extended response questions, terminology questions, process and arrangement questions. The assessment will be conducted online and will have a duration of 2 hours and 15 minutes for writing, with an additional 15 minutes allocated for reading, downloading, and uploading materials.

This assessment is worth 20% of your final grade. It is designed to gauge your progress approximately halfway through the term and to help you identify areas of strength and areas for improvement within the unit. Additionally, it aims to familiarize you with the structure and types of questions you can expect on the final exam.

Please note that this written assessment will be accessible online from 9 am, on Sunday 14th April, and it will remain open for 24h and 30 min to complete this assessment once commenced.

Mode: Online submission

Format: The assessment may include (but not be limited to) some short answer questions & some with a more extended type of response, terminology questions, process and arrangement questions & case study/ies. Marks will range from 1-2 marks for short answer questions and 4-5 marks where more detailed information will be required.

A detailed marking criteria and marks allocated for each question will be provided with this assessment. You will be provided with support and examples of the types of questions you are likely to encountered in this assessment during your scheduled classes; this will assist you in learning and understanding the expectations of this assessment. You are therefore strongly encouraged to regularly attend and actively participate in the weekly scheduled classes, engage and ask
questions where you are uncertain and make sure you come prepared for each class by having reviewed any pre-class learning material. If you still have questions or areas you do not understand following each weekly tutorial class you will be encouraged to address these promptly by posting your questions on the Discussion forum and engaging in discussion on this/these topics with fellow students and academics, and the Unit coordinators. Doing this will ensure you 'arrive' to this.

Task: As a senior scientist in a pharmaceutical company, you have been assigned to a project involving the virtual mass production of a protein implicated in several diseases. All you have been given is the name and the molecular weight of the protein in kilo Dalton (kDa). To achieve this, you need to use the protein and nucleotides sequences, obtained from NCBI, to clone the gene encoding the protein of interest into an expression plasmid DNA vector and mass produce it in a host. You have been assigned to write a report in which you must outline the strategies and methodologies that you recommend. 

Before you go any further, please go to the subject Moodle site and watch an instructions video provided for Assessment 2. In addition, an example of a report is provided in the subject Moodle site to help you with the format of the assignment. Watch this video: https://www.youtube.com/watch?v=QLcmEqBayr0&t=143s  and this one:   https://www.ncbi.nlm.nih.gov/

Project Overview and Guidelines:

Topic Selection: Choose a protein from a list of proteins provided in the subject Moodle site, under assessment 2. There are 17 available topics, and each topic can accommodate a maximum of two students. The selection process operates on a first-come, first-served basis. Once a topic is chosen by two students, it will no longer be available for selection. In that case, please choose another topic from the remaining options.  Please note that you are required to visit the subject Moodle site and select a topic from a list of proteins provided (under assessment 2) by the end Friday, March 22nd, 5:00 PM.  Prepare your report according to the following sub-headings and order: 

1. Aims and Objectives: The goal is to virtually mass-produce the target protein using efficient strategies and methodologies. Prepare your task in accordance with the following subheadings.
2. Introduction (Literature Review): Use PubMed to conduct a literature review on your target protein. Write an introduction focusing on various aspects, including the protein's structure, molecular weight (in kDa), function, disease associations, amino acid and nucleotides sequences, production methodologies etc.  limit the introduction to a maximum of 500 words.  
3. Go to NCBI to determine the protein and the nucleotides sequence of your protein in FASTA format.
4. Identify the open reading frame (ORF) of your gene (start codon is ATG and the stop codon could be any of these three codons: TAG, TAA, or TGA.
5. Select PCR primers either independently or through NCBI's primer design tool. These primers aim to amplify and clone the gene responsible for encoding the protein of interest. Upon determining the forward and reverse primer sets, include them in your report.
6. Restriction Enzyme analysis: Construct a restriction enzyme map of your nucleotides sequence so that we can design a cloning strategy: (https://www.neb.com/en-au/tools-and-resources/interactive-tools).
7. Results: Present the amino acid sequence of the protein, the nucleotide sequence of the gene encoding it, restriction enzyme map of the gene to be cloned, and the forward and reverse PCR primers you have designed. 
8. Cloning Strategy: Outline your cloning strategy based on the chosen expression system, ensuring the efficient insertion of the target gene into the expression plasmid DNA vector. Optimize the vector for high-yield protein expression.
9. Outline the Expression System: Based on the literature review and bioinformatic analysis, choose an appropriate expression system for virtual mass production. Consider factors such as protein solubility, post-translational modifications, and scalability.
10. Host selection: Select a suitable host, either a prokaryotic system such as such as a bacterium or a eucaryotic system such as cell lines, and optimize conditions for protein expression. Evaluate and modify growth media, induction conditions, and harvest timing for maximum yield.
11. Describe the Purification Protocols: Develop robust purification protocols. Prioritize purity, yield, and scalability in the purification process.
12. Outline Quality Control Measures: Implement quality control measures at each stage of the production process. Employ techniques like SDS-PAGE, Western blotting, and mass spectrometry to verify protein identity and purity. Conduct in vitro assays to determine the biological activity of the produced protein. Conduct in vivo studies to determine the protein's serum half-life and toxicity. Conduct pharmacokinetics (PK) and pharmacodynamics (PD) studies.
13. Documentation and Reporting: Document each step of the process, including protocols, results, and any deviations encountered. Provide clear and concise instructions for junior scientists and technical staff to follow. Have your draft read by a peer and check for inconsistencies, typographical errors, and grammar errors, etc. Submit your final draft on the subject Moodle site.
14. Submit online.

Please refer to assessment 2 table of Rubics in the subject Moodle site.