2022/23 Taught Postgraduate Module Catalogue
BIOL5373M Protein Engineering Laboratory Project
15 creditsClass Size: 96
Module manager: Dr Chi Trinh
Taught: Semesters 1 & 2 (Sep to Jun) View Timetable
Year running 2022/23
Pre-requisite qualificationsBSc in Biological Sciences or equivalent
|Bioscience MSc Research Project
This module is not approved as an Elective
Objectives- To provide practical skills training in a range of modern biological experimental techniques principally based around molecular biology.
- To provide training in maintaining laboratory records and in writing scientific research papers.
On completion of this module, students should be able to:
1. demonstrate a high-level understanding of and practical competence in a range of experimental techniques that underpin modern molecular life sciences including: plasmid DNA preparation and quantification; subcloning; PCR; site directed mutagenesis; DNA sequencing; recombinant protein expression; SDS PAGE and western blotting; protein purification and protein analysis;
2. maintain a detailed and accurate laboratory notebook recording the experimental procedures and results obtained during the practical sessions;
3. present and critically analyse the results obtained in the form of a short scientific paper.
This module is an extended practical investigation in the form of a mini-project. The aim is to sub-clone the gfp (green fluorescent protein) coding sequence into an expression vector, express the protein, purify it and then analyse some of its properties. To achieve this, you will work individually through the following experimental stages:
1. Sub-clone the coding sequence of gfp DNA from pET23 into the pET28c protein expression vector (practicals 1-3). Plasmid maps for pET23 and pET28c are included in Figure 1.
2. Transform the plasmid into E. coli DH5a cells and select by plating on LB medium containing kanamycin (antibiotic) followed by direct colony PCR to detect the presence of the gfp insert (practicals 3-4).
3. Generate 1 spectral variant (mutation) of gfp using site-directed mutagenesis and characterize by DNA-sequencing. Overall, this will yield 2 pET28c recombinants, one carrying the wild-type gfp construct and the other carrying (different) mutated gfp constructs (practicals 5-6).
4. Isolate plasmid DNA derived from each of these recombinants and transform into the expression host BL21(DE3) pLysS (practicals 6-7).
5. Induce GFP protein expression (including mutants) using the auto-induction method and assess protein expression by SDS-polyacrylamide gel electrophoresis followed by western blotting, using HisprobeTM-HRP (practicals 8-9).
6. Purify the GFP proteins using Ni-NTA chromatography (practical 10).
7. Analyse the purified proteins by mass spectrometry and fluorometric analysis (this will be done for you and the data returned to you to analyse: practical 11).
|In Course Assessment
|Private study hours
|Total Contact hours
|Total hours (100hr per 10 credits)
Private studyStudents should note that the following information is for guidance only. The actual time required for the various elements will vary between students.
- 2.5hrs preparation per practical: 27.5 hours
- Primer-design activity: 3.5 hours
- Preparation of end of module test: 5 hours
- Preparation of short literature review and research paper: 50 hours
Opportunities for Formative FeedbackThis module will be delivered by laboratory training and experimental practice. You will be required to keep an individual contemporaneous record of procedures and results in the form of a laboratory notebook, which should be available for inspection by the module teaching staff during every practical session. The results of the investigation will be written-up in the form of a short research paper (in the style of FEBS Letters) at the end of the practical.
You are expected to come to each practical having read the protocol and the preparatory (background) reading associated with each practical. This preparatory reading will include some questions intended to assist you in your understanding. These should be completed prior to attending the classes. The demonstrators will go through the answers during your practical session. End of module test questions will be derived from these.
Methods of assessment
|% of formal assessment
|Formative (non-assessed) 800 words literature review on "The properties and uses of GFP".
|End of module test - an online test comprising of a series of short questions and multiple-choice questions.
|Scientific research paper FEBS Letters journal. Presentation and critical analysis of results obtained in the form of a scientific paper.
|Laboratory book assessment
|Total percentage (Assessment Coursework)
Normally resits will be assessed by the same methodology as the first attempt, unless otherwise stated
Reading listThe reading list is available from the Library website
Last updated: 13/10/2022
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