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.

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.

Practical sessions including some bioinformatics.