How does heat shock impact the yeast cycle of life?

How does heat shock impact the yeast cycle of life?

How does this fascinating question came in our mind?

For the final project of the Biosensors month, we decided to focus on…

thermal shock !

In microbiology field, thermal shocks are mainly used for DNA cloning. Cloning is a major technique used in microbiology and molecular biology in order to artificially or naturally multiply DNA, usually a gene, thanks to a micro-organism. It allows a higher expression of these genes useful to protein production. Process includes an alternation of cold and hot temperatures applied on microorganisms culture, as you can see on this video, that compare heat shock and a chemical process. Alteration allows the introduction of a gene inside the cell pursuing cellular division. The gene of interest is therefore transmitted to next generations.

But how does this technique allow the insertion, what happens inside the membrane? It is simple enough: heat fluidifies the proteins and phospholipids presents in the membrane. But shocks can disturb the membrane if the temperature is too high because proteins are denatured i.e they are not functional anymore and phospholipids are disorganized. So a well-balanced temperature has to be choose for the DNA cloning.

Now you know how heat shock works and probably better understand why we wanted to study heat shock effects over micro-organisms membranes for our last week project.  

Figure 1: Here is a small sketch of what you need to know basically on microbiology.

But… How did we answer that question? A beautiful story about yeast and dye

Which cobaye? Baker’s yeast Saccharomyces cerevisiae, a model organism commonly used in biology!

Figure 2: Observation with the microscope of Saccharomyces cerevisiae.

Yeast tolerates temperature between 0 to 55°C but it is a temperature dependant organism and we thought it would therefore be more sensitive to high temperatures. Doing bibliography we found that 50°C was the ideal temperature for shocking yeasts but we then had to figure a way to observe shock impact.

How? a red dye named Phloxine B !

Living yeasts pump out the dye when they are metabolically active (alive) but stay red tinted when they die. We were now able to count the dead cells after the shock and to compare lifespans at ambient and hot temperatures!

Fig. 3: We used S. cerevisiae culture to observe yeast growth and shock them. Growth curve observation is meant to indicate first the right moment to start experiments then the impact of these experiments on yeasts.

Fig. 4: example of growth curve representative of yeasts concentration. Exponential phase is the time when cells divide rapidly so their membranes are flexible therefore the ideal moment to perform our experiments!

What we finally obtain… Not expected but still a beginning:

            Fig. 5: yeasts we have seen under the microscope.

We observed yeasts under the microscope and saw that after 50°C shock, yeasts had a red halo. Our hypothesis about this phenomena is that heat allows the phloxine B to go into cells easier because yeasts first use its energy to keep  their membranes functional, not explode then to expulse the phloxine B. But only boiled yeast (80°C heat shock) were totally red, this is the positive control, which allow us to verify phloxine B action on dead yeasts.

Fig. 6: positive control i.e experiment whom result is known and prove

that protocol is suitable

Our conclusion on this amazing experiment:

Running out of time, we were not able to continue the growth observation and see heat shocks affect over time. Even if cells began to stay red which allowed us to suppose that heat shock effectively reduces yeasts lifespan, we also can not conclude about long term effect.

You may ask what comes next? Well, doing these experiments again!

During this week, we faced some difficulties and finished running after some more time but we all agreed that if we had to try we would keep the protocol but use a different yeast strain: one that kindly accepts to actually grow!

If you want more informations, just click here:

Figure 2 comes from this place!

Figures 3 and 4 let you think that we are artists but were got with the little help of [a] friend

If you want to discover how cool organisms are yeasts, just go here!

We do not have cool pictures to show you but here you can learn how to create fluorescein, Phloxine B cousin dye! Just remember that phloxine does not need fluorescent filter to be seen