We elected to use the yeast Schizosaccharomyces Pombe as the species of use in our biolaser. S. Pombe is a very common model organism in biological reasearch, and we had an excellent opportunity to learn from bioscientists that are very experienced with growing and manipulating these cells at the Lopez-Aviles research group, so they were a natural choice for us. There were several other reasons for us to pick this organism, however. The first one was after TU Delft reported that cell size was a possible limiting factor for lasing their E. coli cells [3] ; this is something the significantly larger S. Pombe cells would remedy, if this was the case. We also wanted to attempt to implement a biolaser in a new cell type: mammalian cells and bacteria have previously been attempted to be used for biolaser gain mediums, however as far as we can tell it has not been attempted in yeast cells before. And, finally, S. Pombe has rarely been used by iGEM-teams previously, which means the work we do here could help future teams that want to work with the organism, by testing whether existing biological parts made for Saccharomyces Pombe (the most commonly used yeast species used in iGEM) still function in another type of yeast cell.

In addition, we wanted to test the laser on a simpler system, namely a protein solution containing large amounts of sfGFP. This was partially to test the setup, but also to examine how a simple system without the cells would function compared to one containing living organisms. For this, we managed to find a particular type of sfGFP that had been modified by a His-tag, allowing for simple purification.

Superfolder Green Fluorescent Protein (sfGFP) that is, compared to regular GFP, more resistant to denaturation and has improved folding kinetics. [4]

Transgenic E. coli is used to synthesize sfGFP and then purify it, which can be used for proving the concept of our biolaser.

In this procedure French Press can be used for bursting the cell wall membrane, in order to release all the cellular components together with the sfGFP. In this technique, pressure differential is used to achieve this. There is a sample outlet tube through which the cells are dispensed slowly (approx. 15 drops per minute). Before releasing the cells, the internal FPC (French Pressure Cell) pressure is increased together with intracellular pressure. As soon as the cells are dispensed, external pressure drops down to almost atmospheric pressure and the intracellular pressure drops too, but slow enough to make the differential pressure significant enough to burst the cell wall membrane.

In the end, chromatography can be used to purify the sfGFP solution even more. It is a system with prepacked column packages and fraction collector which makes the procedure much more reproducible and easier.