Molecular Biology for Discovery Life Sciences

• Credit value: 30 credits at Level 7
• Convenor: Renos Savva
• Tutors: Renos Savva, Professor Sanjib Bhakta, Claire Bagneris
• Assessment: an e-lab book (20%), specialist booklet (40%), short-answer online test (15%) and individual viva (25%)

Combining advanced level molecular biology theory and laboratory practical, this module will cover workflows typical in the early stages of therapeutics research when reliable protocols for obtaining a drug target are paramount. Alongside these parallel learning opportunities, there will be a focus on proficiency with industry-standard electronic lab books and an experience of teamwork around a specialist scientific publishing challenge in which multiple aspects of the publication process will be experienced by all team members. The focus will be on state-of-the-art approaches to drug discovery and other therapeutics centred upon infectious diseases.

Via a combination of on-demand video lectures, interactive seminar discussion, tutorials and laboratory practical classes, learning will be assessed via theory tests in multiple modalities for a thorough learning experience.

Indicative module syllabus

Tutorials

• The E-lab book: Benchling
• The specialist booklet: tasks, milestones and deliverables

Lectures and interactive seminars

• Gene expression circuits in recombinant systems
• Primer design for PCR methods, including for aims such as restriction-ligation and Gibson Assembly
• Bioinformatics for molecular cloning
• Benchling for molecular cloning workflow design and record keeping
• Background vs target in molecular cloning; blue-white screening vs colony PCR
• Quality control of target clones: restriction analysis and sequence validation
• DNA purification methods
• Bacterial transformation by plasmids
• Bacterial recombinant expression
• The T7 recombinant expression system
• Bacterial cell lysis and partitioning
• Protein fusion tags
• Chromatography techniques for protein purification
• SDS-PAGE and Western Blotting
• Small molecule drugs and biologics
• The drug discovery process and approvals pipeline
• Drug repurposing
• Viruses and antivirals
• Microbes, and antimicrobial resistance
• Therapeutic advances in cancer indications

Practicals and tutorials

• PCR a gene (with phosphorylated primers)
• Digest a vector (with blunt end restriction enzyme releasing β-galactosidase gene and phosphatase)
• Agarose gel electrophoresis, DNA (PCR and vector) clean-up, DNA concentration measurement, overlap extension PCR
• DNA purification from agarose gel, DNA concentration measurement, DNA ligation, bacterial transformation
• Plasmid miniprep, Site Directed Mutagenesis via iPCR to introduce a strep-tag-II
• Colony PCR, plasmid miniprep, agarose gel electrophoresis of colony PCR, buffer making for protein purification
• Cell lysis from pre-grown small-scale mutant expression pellets, Strep-tag-II affinity chromatography using mini-spin columns
• SDS-PAGE (electrophoresis), protein concentration by UV, biochemical properties analysis using Benchling, Thermofluor assay +/- drug
• Thermofluor data analysis; specialist booklet tutorials in groups

By the end of this module, you will be able to:

• use the Benchling online lab book and its bioinformatics modules, and similar external online resources (primer design, sequence analysis, plasmid mapping and construct design), to design and record experiments in a recombinant molecular biology workflow
• design and assemble ad hoc combinations of DNA precursors in silico and then in the laboratory, to build recombinant plasmid constructs capable of directing expression of a recombinant protein
• categorise different elements of genetic circuits and their functions in optimising expression of recombinant proteins
• apply standard protocols in DNA purification: (a) from small-scale cultures of E.coli to obtain pure samples of plasmid DNA; (b) from reaction mixtures to remove reactants other than DNA, and (c) from excised fragments of agarose gel after electrophoretic separation of samples
• apply standard protocols in lysis, partition and affinity protein purification from small-scale cultures of E.coli to obtain pure samples of Strep-tag-II tagged recombinant protein
• perform and interpret diagnostic and preparative gels and blots in the lab, typical of molecular biology workflows
• perform and interpret biophysical analyses of a protein with and without bound drugs using the Thermofluor assay
• define the problems being faced in drug discovery with interpretation and summary of infectious diseases
• explain and illustrate via synthesis of written and graphical media a wider appreciation of therapeutic approaches across a variety of healthcare indications
• define and state relevant information recalled as relevant to the material encompassed in the module, namely recombinant methods, molecular biology as a tool for drug discovery, and therapeutic approaches.