SU Course: Methods in Molecular Life Sciences Part – 2

Today I will talk about the wet lab aspect of the Practicals of the methods course.

2. Lab Practicals

The main aim of the lab exercise was to generate mutant strains of the Saccharomyces cerevisiae and look for the mutations in genes involved in Lysine production  pathway using Illumina sequencing.

1. Experimentation and Data generation – Wet lab

a. Yeast Culturing

We started by culturing yeast in plates from the culture flasks by using different OD to get a good growth that can be used for further experimentation. Saccharomyces cerevisiae or commonly known as Yeast takes about 2 days for good growth. Thus, after streaking on the YAPD (rich media) we let them grow for about 2 days.

b. Clonal Purification

The yeast cells were then clonally purified on nitrogen-poor media that contained high levels of α-Aminoadipate and lysine. This is an intermediate from the Lysine production pathway. However, downstream synthesis product derived from this intermediate is shown to cause the growth inhibition on nitrogen poor media. Thus, if cells do not have the 3 important lysine genes (LYS2, LYS5, LYS14) then the conversion of α-Aminoadipate to the toxic intermediate does not take place, allowing the cells to grow.

Selecting clones for plating and obtaining genetically purified (identical) colonies

Thus, we clonally select cells that have mutation in these Lysine genes or so called auxotrophic for Lysine.

c. Validation of auxotrophy

To verify that the cells thus selected actually have the mutations in the lysine genes, they are replica plated on Lysine free media to see for no growth as opposed to lysine rich media.

The third column shows a mutant strain, as it has no growth in Lysine deficient media

d. Complementation test

Plate containing the auxotrophic mutants was crossed with the tester strains (strains that are known to have mutations in LYS2, LYS5 and LYS14 genes) by replica plating.  This test is used to determine experimentally the specific gene in which each mutant has a mutation. This observation is used to obtain preliminary information if it is worth sequencing the sample and where to expect the mutation while analysing sequencing data. Good samples were selected and used to perform Illumina sequencing.

Replica plating for complementation test (crossing tester strains with the mutants from the velvet to the plate)