Biochemistry at Le Loft.
The workshops described on this page are for A Level or IB students. They assume a familiarity with laboratory procedure and basic techniques. If you are looking for our introductory workshops, please follow this link.
The Growth and Reproduction of Saccharomyces spp.
This was once our most challenging workshop series, but it's recently been tipped off its perch by our 'Purification of catalase'. workshop. Between them, these two workshops will span the two years of a college course.
The Growth and Reproduction of Saccharomyces spp. is broken down into a number of modules; each module requires a '3x' block of tuitions, i.e. 4 x 3 hours per block. This is a demanding and complex workshop; it is recommended that it is undertaken as a fortnightly 3hr two-to-one. This is an ideal project for a pair of high-attainment students who wish to boost their scientific practice to compete effectively with those of international students.
(sessions 1-4 of 12).
Basic Microscopy and Biochemistry: Cell counts, Buffers, Liquid and Solid Media. Use of the balance and pH meter. Aseptic Technique and Plating.
Reproduction by binary fission.
Reproduction by budding.
(sessions 5-8 of 12).
Growth Curves: use of the incubator, cell counts using the haemocytometer, use of the centrifuge. Literature searches, spreadsheet techniques and research methodology.
Saccharomyces spp. Growth Curves I: Introduction.
Saccharomyces spp. Growth Curves II: The Early Phases.
Saccharomyces spp. Growth Curves III: The Late Phases.
(sessions 9-12 of 12).
Experimental Design: hypothesis testing, confidence intervals, f- and t-testing. Advanced spreadsheet techniques: primary and secondary axes, log vs.linear scales, error bars. Formulation of media and buffers. Viable cell counts and plating efficiencies. Innocula.
Saccharomyces spp. Growth Curves IV: The Exponential Phase.
Saccharomyces spp. Growth Curves V: Media composition and population doubling times.
Module 4 (Optional, sessions 13-17 of 17).
Basic molecular biology. Glycolysis, Fermentation, Respiration. Genes and Alleles. Enzymes: kinetics and isoforms. Transcrition factors, promotors. Protein kinase A. Diauxic growth in yeasts.
spp. Growth Curves VI: Catabolite repression.
The purification of catalase.
This workshop was developed using lamb's liver as the source of the catalase. This may have ethical implications for some of our students and so we offer this workshop as it was developed i.e. using lamb's liver, or in the form of a research project using a suitable vegetable tissue. IB students take note...
Term 1: Extraction.
(i) make up extraction, dialysis and assay buffers; creation of crude tissue homogenate;
(ii) activity of catalase standard: redox and back titration;
(iii) catalase activity of raw homogenate.
(iv) protein concentration of raw homogenate; Beer-Lambert law.
Term 2: Salting out and dialysis.
(i) ammonium sulphate fractionation: 50% 'cut';
(ii) dialysis: F1 and F2;
(iii) catalase activity of F1 and F2;
(iv) relative activity of F1 and F2; Lineweaver-Burk plots
(v) paper electrophoresis; image analysis.
Term 3: Salting out: second cut
(i) second cut and dialysis;
(ii) catalase activity of F3 and F4;
(iii) gel filtration;
(iv) specific activity of fractions.
Further work: writing up.
The student is encouraged to write up this project in the form of a formal research paper. This will require a working knowledge of the format of a 'paper'. To this very specific end, we encourage all our senior students to engage fully with our 'Journal Club' tutorials.
Journal Club Tutorials
These tutorials form a regular part of the activities undertaken as part of these workshops.