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Team:Freiburg/Results/Light Boxes
From 2014.igem.org
Our Light Boxes
One main principle of The AcCELLerator includes the illumination of our cells. This means we were in need of highly functional and easy to use light boxes.
Last year's iGEM Team of Freiburg already examined the minimum requirements for a light box and designed an easy approach for building your own. Based on their ideas we developed our ideas for constructing light boxes which more sufficiently suited our own needs.
Each light system works on its optimum with a highly specific wave length, which ensures best activation or inactivation efficiencies. Therefore we merchandised diodes with 660nm and 740nm (Roithner Lasertechnik Germany, LED740-01AU/ LED660N-03) to provide best results and created an easy to replicate circuit allowing straightforward operability.
Fig. 1: Electronic circuit (2 diodes + 1 resistor wired parallel)
Fig. 2: Functional diodes connected according our electronic circuit
Those electronic circuits can be combined freely, permitting us to regulate the size of the light box more easily. This way we build a red and a far red light box which easily fit three cell culture plates at a time for simultaneous experiments. Our boxes are made of simple fabrics from your local DIY-store, consisting of PVC plates and acrylic glass glued together and sealed. To ensure equally emitted light on any cell and reduction of the light intensity we also added the intensity decrease units, recommended by last year’s team of Freiburg (frosted glass, clear foil or white paper).
Fig. 3: Red Light Box
Fig. 4: Far Red Light Box
Establishing our desired conditions we were interested in characterizing our red light boxes and tested the wave length of the build-in diodes by measuring the light spectrum. Especially for the red light activation system we can guarantee the optimal wave length, which is very important for us. The wave length of the far-red diodes is slightly shifted from the optimum but still sufficient for our experiments. We also measured the light intensities of the diodes and evaluated for the 660 nm diodes a value of 110 µmol/m²s, which is toxic for our cell lines. By adding the intensity decrease units we could reach a down regulation to 7 µmol/m²s. The 740nm diodes are naturally less intense with 80 µmol/m²s and values down to 6 µmol/m²s could be achieved by using different types of frosted glass.
Fig. 5: Wavelength measurement Light Box 660 nm
Fig. 6: Wavelength measurement Light Box 740 nm
For operating with patterns even less intensities (0.5 µmol/m²s) are recommended [1]. Conducting further experiments we were kindly supported by the Weber’s lab from BIOSS - Centre for Biological Signalling Studies, Freiburg. In using their light boxes we were able to down regulate the light intensity electronically to reach the favored conditions.
In addition to the red light system we also tested the blue light system, which offers simpler completion and higher efficiencies. That means we were also in need of a functional blue light box and tried to improve the uniBOX of the iGEM Team Freiburg 2013 for our needs. We used the identical LED band as last year' team and build it into our own construct.
Fig. 7: Blue Light Box
To ensure the same conditions we measured the light spectrum of our LED band similar to last year’s team and generated an equivalent result with only minor deviations. The blue light intensity in our box was measured 9 µmol/m²s.
Fig. 8: Wavelength measurement white light
Most of our patterning results are based on this blue light system, which meant we needed to improve the lightning conditions even further and created another light box. With this device we were able to reach an almost complete elimination of stray light and were no longer in need of a photo mask. By using this box with an Arduino-microcontroller we can trigger each diode (460 nm) individually via our computer program and can also easily change the light intensity. 100% intensity equals 170 µmol/m²s and best results were achieved with 5 % intensity equaling 6 µmol/m²s.
Fig. 9: New Blue Light Box
Fig. 10: Controlling single diodes
References
- Müller K, Zurbriggen M & Weber W (2014). Control of gene expression using a red- and far red light-responsive bi-stable toggle switch. Nature protocols Vol.9 No. 3
