Team:UiOslo Norway/Description
Lasers
A laser is a device that emits monochromatic light amplificated by stimulated emission of electromagnetic radiation, hence the name (“Light Amplification by Stimulated Emission of Radiation”).
The first laser was built in 1960 by Theodore H. Maiman, based on theoretical work by Charles Hard Townes and Arthur Leonard Schawlow. What makes a laser different from other light sources is the fact that it emits its light coherently. This means that the stream of light will stay narrow over a long distance and can also be focused in a tight spot, like for example for laser cutting or a laser pointer.
A laser consists mainly of five parts: the gain medium, the laser pumping energy, the high reflector, an output coupler and a laser beam. The gain medium is a material which allows light to amplify. This is usually located in an optical cavity. What this means is that there is a mirror on either side of the medium, in order to make the light bounce back and forth. This amplifies the light, as it passes through the gain medium each time. One of the mirrors is usually not 100% reflective, since there will a small output (the laser beam). For anything to happen within the optical cavity we need a power source that allows the gain medium to amplify the light. This energy is supplied through a process called pumping and is usually either light or an electrical current.
When the laser was invented in 1960, it was initially described as "a solution without a problem"(citation?), as it wasn't immediately obvious what it would be useful for. In retrospect we know that this is one of the most valuable inventions in the last 100 years as lasers are used for a wide variety of things; surgery, measurements, information reading and processing, industrial purposes and weapons. They also have a central role in a lot of popular culture and is loved by nerds all over the world.
Enter the biolaser
In 2011, a paper was published in Nature Photonics [1] on using a single mammalian cell as the gain medium for a biolaser, describing the coherence and amplification seen in non-biological lasers. There have been several other cases of work on biolasers afterwards [2], including work done by 2016's winner of the new application track at iGEM. All of these projects have mentioned the potential of using the amplification effect of a biolaser to improve sensitivity of existing measurement methods, as the amplification effect of a laser could make for very sensitive changes in the concentration of fluorescent material. This is a potential we wanted to investigate. One of the great limitations of science is the sensitivity of the methods of measurement, and being able to improve this would surely be a boon to cell biology in the future.
LaCell - Project Plan
The basic plan for our project is two-pronged:
-Creating a functional biolaser setup by using a fluorescent protein solution as the gain medium for a laser
- Apply said biolaser setup and use living cells containing fluorescent protein as the gain medium
The idea is to first make a functional proof-of-concept on the biolaser and assert how energy applied changes the output of a biolaser. If this is a non-linear relationship, it would indicate potential of a biolaser as a more sensitive method of measurement.
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.
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.
References:
1 - Nature Photonics 5, 406-410 2011: Single-cell Biological Lasers, Malthe C. Gathers & Seok Hyun Yun - DOI:10.1038/nphoton.2011.99
2 - Science Advances 19 Aug 2016: An exciton-polariton laser based on biologically produced fluorescent protein, Dietrich et al - DOI: 10.1126/sciadv.1600666
4 - PLoS ONE, 1-7 2008: Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control Fisher, A. C., & DeLisa, M. P
5 BioMol.net. (2017, July 30). Retrieved from Protein Extinction Coefficient Calculator: http://www.biomol.net/en/tools/proteinextinction.htm
6 iGEM. (2017, October 30). Help:Protocols/Transformation. Retrieved from Registry of Standard Biological Parts: http://parts.igem.org/Help:Protocols/Transformation
