Molecular Biology for Discovery Life Sciences

• Credit value: 30 credits at Level 7
• Convenor: Professor Katherine Thompson
• Assessment: short-answer questions (10%), two sets of lab book entries (30% and 35%) and a poster presentation (25%)

Combining advanced-level molecular biology theory and laboratory practicals, in this module we 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, you will become proficient with industry-standard electronic lab books and engage in 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 syllabus

Tutorial

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

Lecture and interactive seminar

• 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
• Microbes, and antimicrobial resistance
• Therapeutic advances in cancer indications

Practical and tutorial

• 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 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.