Team:Freiburg/Project/unibox

uniBOX - A lightbox built from lego!

Idea

Light inducible systems are more and more used in iGEM and synthetic biology in general. Team Munich for example uses a light inducible killswitch, while Shanghai uses light-controlled expression systems. Even our uniCAS gene regulation system is controllable via light. Therefore we thought of an easy and cheap way to build a light box to illuminate our cells with a certain wavelength. But construction of such a light box can be hard because there are certain problems you have to face like dimout but still enough oxygen supply or illumination with the right wavelength. Our so called uniBOX can be build by using common things like Lego bricks, glass, foil and LEDs. We were able to show that light systems can be controlled efficiently and we now provide a do-it-yourself manual for building your own uniBOX for less than 60 €!

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) (Müller et al., 2013). 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 an mCherry reporter gene. Therefore after an illumination with 660 nm 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 Müller et al., 2013.

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

Results and Discussion

Testing the uniBOX with a good characterized red - light system

At first we measured the spectrum of our LED light chain bought in a common home improvement store (Figure 1). As this LED light chain is built on a RGB color space three different LEDs were used: Blue, Green and Red. For the measurement of the spectrum the LED light chain was set to white light. Three peaks were measured. One at 459.33 nm corresponding to the blue LED, one at 519.59 nm corresponding to the green LED and one at 632.6 nm corresponding to the red LED. It is obvious that the red LED with its peak has not the optimal condition for performing light experiments. Therefore we had to do some experiments to test whether the TetR-VP16 activation system will also be inducible at 632 nm instead of 660 nm.

Figure 1: Spectrum of the uniBOX LED light chain The LED light chain was set to white light and the spectra of all three RBG LEDs were measured.

As proof of principal for an activation at 632 nm we illuminated a 10 cm culture dish containing CHO-K1 cells. Another plate was kept in dark for control. The results can bee seen in figure 2. For both plates the same exposure time was used. Cells illuminated with 630 nm red light for 1 h show a strong mCherry signal compared to the control plate. The weak fluorescence of the control plate is due to the leakiness of the CMV minimal promotor.

Figure 2: Red light induced mCherry expression (left) compared to a dark controle (right). CHO-K1 cells were either illuminated with red light in the uniBOX (left) or kept completely in dark (right). The rasters seen in the left picture are due to the scanning of the cells via a fluorescent microscope and due of the weak mCherry signal.

Spatio-temporal gene expression

The best results for spatio-temporal gene expression were achieved by illuminating CHO-K1 cells for 1 h with an intensity of about 0.02 - 0.1 µE using red LEDs. In general a 10 cm culture dish with an even and dense cell distribution was illuminated through an edit format. Therefore only small parts of the plate were covered in light resulting in a production of mCherry. This method allowed us to write the word "iGEM" (Figure 3), to draw a heart (Figure 4) and even to display a rudimental iGEM logo (Figure 5) within the cells.

Figure 3: Red light driven mCherry production in CHO-K1 cells using spatio-temporal gene expression 3.5x10^6 cells were seeded and transfected with tetR-pif, vp16-phyB and mCherry reporter. The illumination was proceeded at 0.5 µE for 1 h through an "iGEM" edit format (top picture). The right picture is showing the fluorescence of mCherry and was proceeded using imageJ with a Gaussian Blur(1.0) and by adjusting intensity and contrast. The word "iGEM" is readable within the cells.

Figure 4: Red light driven mCherry production in CHO-K1 cells using spatio-temporal gene expression 3.5x10^6 cells were seeded and transfected with tetR-pif, vp16-phyB and mcherry reporter. The illumination was proceeded at 0.5 µE for 1 h through a "heart" edit format (left picture). The right picture is showing the fluorescence of mCherry and was proceeded using imageJ with a Gaussian Blur(1.0) and by adjusting intensity and contrast. A heart is visible within the cells.

Figure 5: Red light driven mCherry production in CHO-K1 cells using spatio-temporal gene expression 3.5x10^6 cells were seeded and transfected with tetR-pif, vp16-phyB and mcherry reporter. The illumination was proceeded at 0.5 µE for 1 h through a iGEM logo edit format (left picture). The right picture is showing the fluorescence of mCherry and was proceeded using imageJ with a Gaussian Blur(1.0) and by adjusting intensity and contrast. The iGEM logo is visible within the cells.

Figure 3 -5 is only a collection of our best pictures. The picture quality strongly depends on the density of cells as well as on the efficiency of the transfection. In either case a failure would result in a diffuse resolution. Another important factor is the illumination time and the scattering of light. Both has to be optimized empirically. In our experiments we removed the lid of the culture dish and covered the top of the uniBOX with a black rag to minimize backscattering.

Summary and Outlook

We showed that our uniBOX is able to efficiently activate gene expression using red light. Therefore the iGEM team Freiburg 2013 provides an easy and cost efficient protocol to perform light experiments. Furthermore with help of Konrad Müller and Hannes Beyer we were able to show spatio-temporal gene expression using the uniBOX. Spatio-temporal gene expression itself is a big step forward in the direction of tissue engineering. Already simple structures as shown with the iGEM logo can be translated into gene expression. With modern equipment like a red laser in the right focus plane it should be possible to minimize light scattering and activate even cells. Together with our uniCAS tool that is able to target multiple genes at once on different light stimuli different pathways in different cells could be activated or repressed.

References