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BSc Molecular Biology

Year of entry: 2021

Course unit details:
Molecular Biology RSM

Unit code BIOL20352
Credit rating 10
Unit level Level 2
Teaching period(s) Semester 2
Offered by School of Biological Sciences
Available as a free choice unit? No

Overview

This Research Skills Module is an example of research-informed teaching and will develop your experimental design, report writing and practical skills. You will be introduced to modern molecular biology research techniques in a laboratory-based project in which you will investigate the N-linked glycosylation of proteins from the bacterial pathogen Campylobacter jejuni. You will use basic bioinformatics tools to identify putative N-linked glycoproteins and then test your hypotheses experimentally. This will involve PCR amplification of the corresponding gene and cloning into a suitable plasmid that will allow you to test its glycosylation status. Experiments will be performed in glycocompetent Escherichia coli and involve Western blotting to demonstrate glycosylation and site-directed mutagenesis to identify the specific amino acid residue(s) glycosylated. Successful experiments will identify novel N-linked glycoproteins.

 

Pre/co-requisites

This unit is compulsory for Molecular Biology honours students and may be selected by Biology, Biomedical Science or Biotechnology students. It is important that students not registered on the Molecular Biology Degree programme consult their Programme Director or the Unit Coordinator.

 

Aims

The unit aims to increase the students understanding of the following:

 

1. specific skills associated with molecular biology

a) the use of bioinformatics tools to develop hypotheses regarding protein glycosylation

b) design of primers for polymerase chain reaction amplification

d) polymerase chain reaction amplification of DNA

e) agarose gel electrophoresis of DNA

e) plasmid cloning methods and subsequent verification

f)  plasmid transformation into Escherichia coli

g) production of protein lysates and analysis of proteins by western blotting

h) the use of site-directed mutagenesis to specifically alter amino acid sequence of proteins

 

2. how to design and plan experiments and work independently.

3. how to analyse, interpret and record data.

4. how to present data in a research paper format.

 

 

Learning outcomes

Students will develop an understanding of the basis of several commonly used molecular biology techniques and have the confidence to use these in a practical setting to identify novel Campylobacter jejuni N-linked glycoproteins and the sequence requirements for glycosylation. Students will appreciate the need for good experimental design and laboratory practice.

 

Syllabus

In the Molecular Biology RSM students learn specific molecular techniques and strategies used to investigate gene function. Weeks 1 and 2 of the RSM are carried out in conjunction with the Microbiology RSM. Weeks 3 and 4 comprise only those students taking the Molecular Biology RSM.

 

Students work in pairs.

 

Week 1 Putative glycoprotein encoding genes will be PCR amplified from supplied Campylobacter jejuni genomic DNA and verified by agarose gel electrophoresis. PCR products will be directly ligated into suitable vector for expression in Escherichia coli using robust In-Fusion cloning methods. Following transformation PCR from E. coli colonies will be used to verify cloning and colonies will be inoculated into LB broth for plasmid minipreps. These will be digested and analysed by agarose gel electrophoresis. These approaches will provide theoretical understanding and practical experience of a number of molecular biology techniques. Students will also use bioinformatics to identify putative glycoproteins and will be asked to consider the experimental design aspects of the RSM.

 

Week 2. In week 2, students will test their hypothesis that the genes cloned in week 1 encode glycoproteins. This will involve determining the glycosylation status of the putative glycoproteins by Western blotting experiments using N-linked glycan specific antiserum. Putative glycoprotein-encoding genes will be expressed from vectors in glycocompetent E. coli strains that have complete functional C. jejuni N-linked glycosylation systems encoded on a second vector.

 

Week 3 In this final section of the RSM, students will investigate specific sequence requirements for N-linked protein glycosylation. In week 3 this will involve using site directed mutagenesis to change asparagine residues within the N-linked glycosylation sequons of glycoproteins to alanine. The glycosylation status of these variants will then be tested by Western blotting. These and similar experiments in week 4 will be carried out in E. coli as described in week 2.

 

Week 4 In the final week students will be asked to design their own experiments to make specific site directed mutants in their proteins that might influence N-linked glycosylation. They will be guided in this by supporting material but will essentially make their own choice as to what amino acid sequence changes they attempt to make. This could include changes within existing N-glycosylation sequons beyond the asparagine residue or introducing further N-glycosylation sites. Primer design will be carried out in week 3 and experiments carried out in week 4.

 

 

Teaching and learning methods

Practical sessions including some bioinformatics.

 

Transferable skills and personal qualities

 

Category of outcome

Students will:

Knowledge and understanding

Understand the basis of several commonly used molecular biology techniques

 

Intellectual skills

Be able to design experiments to test a hypothesis and to analyse and interpret the data obtained.

 

Practical skills

Develop skills in several experimental methodologies relevant to molecular biology

Transferable skills and personal qualities

work as a team

problem solve

present data

project manage

time manage

 

Employability skills

Analytical skills
Students must describe and analyse the results of PCR and cloning, transformation, Western blots and SDM experiments. These practical approaches are underpinned by bioinformatic analyses.
Group/team working
Students work at the bench in pairs.
Innovation/creativity
Students are asked to design PCR primers based on the bioinformatic analysis of a gene of interest and also design primers for site directed mutagenesis experiments.
Problem solving
If students do not get the 'expected' results then they will employ problem solving skills to identify what might explain this. Primer design aspects (above) also incorporate problem solving.
Research
Students carry out a structured four-week mini project involving hypothesis generation (identification of putative glycoproteins) and subsequent testing. If successful students will add to existing knowledge and there is even potential for publication. Student will learn theoretical and practical aspects of a number of molecular biology techniques.
Written communication
Students will be required to, and be advised on how to keep a thorough record of their experiments in a lab book. Weekly short answer questions will test their knowledge and understanding. Students will write up their results in the form of a short scientific paper.
Other
Students gain confidence working in a laboratory, making solutions, researching protocols, following protocols, setting up experiments, and analysing the results. Importantly students will have the opportunity to generate novel data and see how their experiments will add to scientific knowledge and consider how this might be disseminated.

Assessment methods

Method Weight
Other 20%
Written assignment (inc essay) 50%
Set exercise 30%

Students maintain a lab book and this will be reviewed and feedback provided. There will be no limit on length but students are expected to concisely but clearly and thoroughly describe the experimental work. Hard copy of lab books returned to students with feedback within 15 work days of submission. 20%

 

A weekly set of five short answer questions will be provided each Thursday testing students understanding of the week’s work. Feedback provided as annotated assessment sheets returned within 15 work days of submission although we will attempt to provide rapid feedback that can be used to improve subsequent assessments where possible  30%

 

The RSM will be written up as a short research publication no longer than 6 pages in length, submitted in Blackboard and feedback provided via Grademark within 15 work days of submission. 50%

 

 

Feedback methods

In addition to feedback on assessments, informal formative feedback will be provided in the laboratory by staff and demonstrators throughout the 4-week practical.

 

Recommended reading

Nothaft H & Szymanski CM (2010) Protein glycosylation in bacteria: sweeter than ever. Nature Reviews Microbiology (8) 765–778.

For Information and advice on Link2Lists reading list software, see:

http://www.library.manchester.ac.uk/academicsupport/informationandadviceonlink2listsreadinglistsoftware/

 

 

Study hours

Scheduled activity hours
Practical classes & workshops 60
Independent study hours
Independent study 100

Teaching staff

Staff member Role
James Linton Unit coordinator

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