Team:Freiburg/Project/unibox

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uniBOX - A lightbox build from lego!

What you need

Using the light box and performing light experiments is easy and can be done in every lab. All you need for your experiments is the following:

• The uniBOX (see construction)
• A common box to keep the samples in dark
• A room that can be made impervious to light (even a store room would serve perfectly well)
• An incubator (also impervious to light in the optimal case)
• 2 LED light chains (one for the light box and one for the "dark" room to get your save light conditions)
• A radiometer to measure light intensities

Principles of light experiments

In principle every light experiment works similar. All you have to do is to proceed your samples under safe light in the dark room. Otherwise it is indispensable to keep your samples in the uniBOX or dark box to protect them from sunlight and artificial light. Even inside of the incubator this protection is important. Safe light are light conditions that do not affect your light experiments. That means if you are doing a red light experiment it is important that the safelight does not include wavelengths form 600 to 700 nm. Therefore the safe light conditions for red light can be green light or blue light. As green light contains less energy it is probably the best choice. Nearly every common green LED can be used to produce safe green light.
For blue light experiments red LEDs can be used to produce safe light conditions.

Set up the light box

For some light experiments it may be of great importance to regulate the light intensity. Using the uniBOX you can do this by adding layers of baking paper between the LEDs and your samples. For very small light intensities you can use normal paper instead of baking paper. The appropriate place to add these papers was described here . The light intensity in general can be measured using a radiometer.

To test our light Box (uniBOX) we performed a spatio-temporal gene expression experiment by using a red light inducible Tet-Off system (TetR fused to PIF and VP16 fused to PhyB) generously provided by Konrad Müller. To get more information about the general functionality of the PhyB - PIF red light system klick here. TetR is able to bind to a TetO DNA target side that was cloned in front of a CMV minimal promotor driving a mCherry reporter gene. Therefore after an illumination with 660nm the gene activation domain VP16 will be brought to TetR resulting in an expression of mCherry. All following experiments were done with chinese hamster ovary cells (CHO-K1 cells).

Method - Spatio-temporal gene expression

This protocoll was adapted from Konrad Müller.

1. 3.5*10^6 CHO-K1 cells were spread in a 10 cm culture dish.
2. After 24 h the cells were transfected with 12 µg of tetR-pif and vp16-phyB and 8 µg of mcherry reporter.
3. A change of culture medium was performed 4 h post transfection.
4. Again culture medium was exchanged after 24 h with culture medium containing 15 µM of PCB under safe green light.
5. After an incubation of 1 h in dark the plate was illuminated with red light at an intensity of 0.02 - 0.5 µE for 15 - 60 min. To gain a spatial resolution different edit formats were used. These edit formats contain of glass masked with black tape containing specific gaps.
6. Thereafter cells were kept in dark for 20 h.
7. To conserve the cells and stop gene expression the cells were washed with 10 ml ice cold PBS (containing Ca++ and Mg++), fixed with 3ml of ice cold 4% Paraformaldehyde (PFA), incubated for 10 min on ice and covered with 3-4 ml of PBS (containing Ca++ and Mg++) after removal of PFA.
8. The cells can now be kept in dark and at 4°C for several days