DEMONSTRATE

OVERVIEW

Figure 1. Our project combined the four elements together: light, sound, odor, color. And the process was divided to three parts: sound to light, light to color, light to odor.

Our project retold the nightingale and the rose [1]using the tool of synthetic biology. In the story, the nightingale song all night under the cold moonlight, stained and scented the flower by its blood. We picked out four elements—sound, light, color and odor from the original story and combined them together. In our project, E. coli needed light and sound as inputs to produce color and odor as outputs. The process was mainly divided into three parts: sound to light, light to color, and light to odor. The light-color and light-odor conversions were achieved with the RGB system[2] which was based on the phage RNAP system as a resource allocator. As for the sound-light conversion, we developed a software that allowed users to upload their own music to generate their unique dynamic pictures, with which people could color and ‘ensoul’ their own roses.

SOUND TO LIGHT

The sound-light conversion was achieved by our software—Orpheus. First of all, users could choose an image file and an audio file stored in their computer. Then Orpheus would graying the inputted image and carefully decoded the inputted audio into wave shapes. After that a series of spots would be generated with corresponding hue and brightness on the image. For hue, Orpheus first decoded sound into wave shapes and analyzed its low, medium and high volume, then generated RGB values with our delicate mapping function. For brightness, Orpheus converted source photos into grayscale images which is in proportion to brightness. The random dropping process, just as the nightingale slowly colored the rose as it warbled in the story, each drop would color some area by the aforementioned method accompanied by the inputted music and finally a beautiful picture was produced.

Figure 2. Some beautiful outcomes of our software. The first one came from a poem. The second one came from wind. The third one came from the song of the birds. The fourth one came from rain.

As shown in the figure and videos, our software could generate beautiful pictures with the music going on. We believe that with this software people could enjoy themselves creating, and wandering at the junction of art and science. Our software is available at SOFTWARE

By using a projector, we could cast the picture we got from our software onto our plates. We also built a hardware to connect the computer, projector, and our plates together. (more information, see HARDWARE

LIGHT TO COLOR

In our project, E. coli needed light and sound as inputs to produce color and odor as outputs. The process was mainly divided into three parts: sound to light, light to color, and light to odor. The light-color and light-odor conversions were achieved with the RGB system[2] which was based on the phage RNAP system as a resource allocator. As for the sound-light conversion, we developed a software that allowed users to upload their own music to generate their unique dynamic pictures, with which people could color and ‘ensoul’ their own roses.

LIGHT TO COLOR

We introduced a RGB system to stain our rose using light. The RGB system mainly consists of four modules: a sensor array, circuits, a resource allocator and actuators.

Figure 3. The structure of the RGB system.

The sensor array combines 3 light sensors, Cph8*, YF1 and CcasR, which can respond to lights of different wavelengths. CcasR can sense and be switched on by green (535nm) light. Cph8* is switched off by red (650nm) light, while YF1 is switched off by blue (470nm) light.

To activate gene expression, the signals from the red- and blue- light sensors need to be inverted, which is done by connecting them to NOT gates in circuits.

The resource allocator which connects the circuits and actuators, is based on a split-RNA polymerase system[3], in which the sigma fragments activated by light sensors can combine with the constitutive expressed non-active ‘core’ fragment to form complete RNA polymerases, then activated the expression of actuators accordingly. For more information on the circuit design, see LIGHT TO COLOR .

The actuators we chose were three kinds of color protein: fluorescent protein, chromoprotein and enzyme which can produce colorful products.

First, to make our rose more colorful, we put forward a concept—mixing colors in cells. Then using tandem expression we performed simple proof of our concept.

Figure 4. The color spectrum built from chromoproteins and their tandem expression products.

Next, we used the RGB system to achieve light-controlled color mix in bacteria, not only to test the function of our system, but to further prove our concept. By replacing actuators with fluorescent proteins and enzymes, we successfully built beautiful color spectrums and created a rose using the colors we created.

Figure 5. The color spectrum built from fluorescent color mixing.

Figure 6. The rose we created by using the colors we mixed.

Figure 7. The color spectrum built from mixing color with enzymes.

Then for more precise control of the color, we explored the relationship between fluorescence intensity and the wavelengths and intensity of input light using ELIASA (microplate reader) and flow cytometry (Figure 10, 11,12). After quantitative analysis, we could predict and control all kinds of colors our bacteria would produce. Our concept of mixing color on the bacteria cells were also proved to be reasonable and provided a convenient way for scientists and artists to create new colors and artworks.

Figure 8. The flow cytometry results shows the distribution of the cells in fluorescence intensity. BV421 represented the blue-fluorescence intensity, while the FITC-A represented the green-fluorescence intensity and Pe-TxR-A represented the red-fluorescence intensity. The horizontal axis shew the fluorescence intensity, while the vertical axis shew the number of bacteria cells.

Figure 9. The spectral response of the complete RGB system. “%maximum induction” is calculated as the fold change of the response divided by the maximum fold change across the spectrum.

Figure 10. The curves showing the relationship between the input light and fluorescent intensity.

By combining the RGB system and our concept of mixing color, we could use the LEDs and projector to produce colorful pictures. As shown in Figure 11-12, the pictures drawn by projector were quite colorful and color gradient could be easily produced, which could hardly be produced using traditional tools of art and science. Furthermore, changing the pictures we projected onto the plates, we could stain our bacteria with more bright color, such as green and yellow as shown in Figure 13. We welcome all iGEMers to create more imaginative artworks just using pictures or sounds in the computer, and our E.coli will help you to create wonderful pictures on the plates. (for more details, please see LIGHT TO COLOR)

Figure 11-12. Pictures produced by casting pictures on the plates by projector. Bright colors and color gradients were easily produced. More imaginative creation using the RGB system are welcomed.

LIGHT TO ODOR

We first tried to use CRISPR/Cas9[4] gene-editing system to knock out the original tnaA gene of E. coli producing smell to prevent E. coli from giving off a nasty and unpleasant smell[5]. And figure 13-14 shew the PCR results of the plasmids and homologous sequence we designed during the process of gene knock-out.

Figure 13. The PCR results of pTarget

Figure 14. The PCR results of Homologous sequence 1, 2, 1+2

Then we applied the RGB system to achieving the light-odor conversion. By using enzymes that produced odor as actuators, we constructed a new plasmid (BBa_K2598062 )and thus could induce the production of the lemon, rain, and flower odor by red, green and blue light, respectively.

Figure 15. The function of the actuators and the wavelengths of input light in RGB system[6]

We then tried to detect our products using HPLC. However, rare previous literature had reported the mobile phase, the temperature, and even the parameter settings of the three molecules, especially geosmin and methyl benzoate. It required a large amount of time and effort for us to discover the right mobile phase and specific detection wavelengths of the products. Considering that our system had been tested by fluorescent proteins and enzymes and it really worked well, we did not spend more time on the HPLC analysis, but we believed that we could do further researches on our products once the conditions and the parameter settings of our products were reported.(More information, see LIGHT TO ODOR)

Demonstrate

To finally demonstrate our project, we constructed a plasmid containing mrfp, gfp, and bmst1 genes as actuators, and transformed it together with pJFR1-3 plasmids into our E. coli, which could be induced to produce red, green colors and flower fragrance by red, green, and blue light, respectively. Figure 14 demonstrated our compare of the picture we cast on the plate using a projector and the picture we drawn by our E.coli. A fragrant and vivid flower was created!

Figure 16. The compare of the picture we cast on the plate and the picture drawn by E. coli. Aluminum foil was used to edge the pictures to make them more clear.

Reference:

[1]Oscar Wilde, 1995, Happy Prince and Other Tales. Everyman's Library, 96

[2] Fernandez-Rodriguez J, Moser F, Song M, et al. Engineering RGB color vision into Escherichia coli[J]. Nature Chemical Biology, 2017, 13(7):706-708.

[3] Segallshapiro T H, Meyer A J, Ellington A D, et al. A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase.[J]. Molecular Systems Biology, 2014, 10(7):742.

[4] Cong L, Zhang F. Genome Engineering Using CRISPR-Cas9 System[J]. Methods in Molecular Biology, 2015, 1239(11):197.

[5] Li G, Young K D. Indole production by the tryptophanase TnaA in Escherichia coli is determined by the amount of exogenous tryptophan[J]. Microbiology-sgm, 2013, 159(2):402-410.

[6] https://2014.igem.org/Team:Paris_Bettencourt