BSc Molecular Biology

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 fin species from the genera Helicobacter and Campylobacter. You will use a range of online bioinformatics tools to identify putative previously uncharacterised N-linked glycoproteins from Campylobacter/Helicobacter species. In the second part of the RSM you will experimentally test the hypothesis that these proteins are indeed N-glycosylated. This will involve PCR amplification of the corresponding gene and cloning into a suitable plasmid that will allow you to express the protein and test whether it is glycosylated using Western blotting. Successful experiments will identify novel Campylobacter or Helicobacter N-linked glycoproteins.

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.

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

1. Specific skills associated with molecular biology

• the use of an array or online bioinformatics tools to develop hypothesis regarding protein glycosylation.
• design of gene-specific chain reaction (PCR) primers and amplification of DNA.
• agarose gel electrophoresis of PCR products d) plasmid construction using state of the art cloning approaches
• plasmid transformation into Escherichia coli
• production of protein lysates and analysis of proteins by Western blotting

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

Students will develop an understanding of an array of online bioinformatics tools. The hypotheses generated will be tested using DNA and protein based molecular biology techniques. The potential for identification of novel Campylobacter or Helicobacter N-linked glycoproteins will allow students insight into how new scientific knowledge emerges in the life sciences from hypotheses to experimental verification. Students will also learn to appreciate how careful experimental design interpretation combined with good laboratory practice is vital for the advance of knowledge.

In the Molecular Biology RSM students learn specific DNA and protein-based laboratory techniques to investigate protein glycosylation status.

Students work in pairs.

Weeks 1 and 2. Students will follow an online Softchalk-based programme of Bioinformatics enabling identification of putative N-linked glycoproteins from bacterial genome sequence data. This involves accessing proteins via UniProt, BLAST analysis, sequence alignment, accessing genome sequence data and analysing via SignalP. In this way students will develop their hypotheses for subsequent experimental testing in weeks 3 and 4.

Week 3. Putative glycoprotein encoding genes identified in weeks 1 and 2 will be PCR amplified and verified by agarose gel electrophoresis. Following purification, PCR products will be ligated into suitable vector for expression in Escherichia coli using state-of-the-art- In-Fusion cloning method. 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 approaches will provide theoretical understanding and practical experiences 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 4. In week 4, students will test their hypothesis that the genes cloned in week 3 encode glycoproteins. This will involve determining the eglycosylation status of the putative glycoproteins by Western blotting experiments using N-linked glycan specific antiserum from vectors in glycocompentent E. coli strains that have complete function C. jejuni N0linked glycosylation systems encoded on a second vector.

Practical sessions including some bioinformatics.

Understand the utility of diverse online bioinformatics tools. Understand the basis of several commonly used DNA and protein-based molecular biology techniques.

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

Be able to apply online bioinformatics tools as appropriate to analyse sequence data including genome sequences and specific proteins. Develop skills in several experimental methodologies relevant to molecular biology.

• Work as part of a team
• Problem solving
• Presenting data
• Project management
• Time management

A weekly set of short answer questions will be provided each Thursday testing students understanding of the previous 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 be used to improve subsequent assessments where possible. (50%) The RSM will be written up as a short research publication no longer than 5 pages in length, submitted in Blackboard and feedback provided via Turnitin within 15 work days of submission (50%).

An initial 1 hour intro session given by the course coordinator will prove the necessary background and discuss the bioinformatics approach - students are encouraged to ask questions here. Each Thursday of weeks 1 and 2 there are drop-in sessions for students to ask questions as they work through the bioinformatics programme. In addition to feedback on assessments, informal formative feedback will be provided in the laboratory by staff and demonstrators throughout the 2 week practical component.

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

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