Template:UCAS-China
One hundred years ago, there was a young boy who was seeking a unique rose for his beloved girl. It was a freezing winter night, all roes had died. After a long time searching in the withered rose bush, the young boy was desperate. Touched by his true love, a nightingale, who heard and understood his wish, sung all night under the cold moonlight. Just before dawn, a rose stained with the blood of the nightingale, bloomed, bright and fragrant.
The writer Oscar Wilde, created a red rose for true love out of the nightengale’s song under moonlight.[1] To Wilde, the unique rose stained with blood of the nightingale is the symbol of love and true art. To us UCAS-China iGEM team, the touching story should be passed down to our generation and explained in a scientific and creative way using the tools of synthetic biology. Furthermore, as Wilde conveyed in the story, the barrier and combination of art and science still remain worthy of discussion, so we also explored in depth the relationship of art and science in the Human Practices section
Our project combines the four elements-music: (the song of the nightingale), light (the moonlight), color (the stained rose) and odor (the rose fragrance). In our project every iGEMer is able to create his/her own with unique soul, together forming our rose forest in the junction of art and science!
Design
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
Firstly, we introduced the 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. 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, 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 (链接light to color).
LIGHT TO COLOR 2
The actuators we chose were three kinds of color protein: fluorescent protein, chromoprotein and enzyme which can produce colorful products. Although more and more fluorescent proteins and chromoproteins are edited to generate more and more colors, the number of colors produced by organisms is still limited by the number of the kinds of proteins. Once a new color is needed, researchers have to modify the chromophores of the proteins, which takes much time and effort.
So how do we create more colors in a reasonable and convenient way? Here, we put forward a new concept—mixing color into bacterial cells! Unlike the mix of different bacterial cells which produce different colors as the previous iGEM teams have done[4], we used tandem expression and RGB system to control the ratios of the expression of different colors in bacterial cells, to achieve mixing color in bacterial cells, and stain our roses with more bright colors. More information- see (Light to color)
We built hardware to generate lights with smooth distribution on the plates and controlled intensity, to activate our system to produce predictable colors. Our final hardware is designed and built to connect computers with our bacteria on the plates, with the help of a projector, to achieve the interaction between users and our E.coli. More information see (hardware)
LIGHT TO ODOR
Our roses are colorful now, but only with added fragrance can the rose bloom more vivid, appealing and with a soul as a real rose. So this is our second part, light to odor, making our roses more soulful and real. We first tried to use CRISPR/Cas9 gene-editing system[5] to knock out the original gene of E. coli producing smell to prevent E. coli from giving off a nasty and unpleasant smell. Then based on the light-control system, we changed the actuators with genes which could produce various kinds of odors.
The inherent unpleasant odor of E. coli comes from indole produced naturally in the cells’ metabolic process. In the L-tryptophan degradation pathway, Tryptophanase, encoded by tnaA gene[6], degraded L-tryptophan of indole, which produces the odor in high concentration. (The enzyme needs Co-factor: pyridoxal 5'-phosphate)
L-tryptophan + H2O = indole + pyruvate + NH3.
We first tried to knock out the tnaA gene in the genome of E. coli, then we replaced the actuators in the RGB system with genes which could produce various kinds of fragrance. We devoted our effort to introducing diversified odor to make our roses not only vivid ones with traditional flower fragrance, but unique ones with more kinds of odor like lemon and rain[7]. Thus we can use light to control the fragrance of E.coli, and the function of our system was tested by HPLC. (see Light to odor)
SOUND TO LIGHT
We could paint a colorful and fragrant rose with light , but it was sound that brought a unique soul to the rose. To create his/her rose with a unique soul, we developed an online software—Orpheus—to convert music into unique colorful pictures. The users could choose their music or their own voice, and the pictures that they want to ensoul, to color the pictures with random colorful dots. When the music was input, after a specified time, the sound waves will be read and the amplitude and frequency of the music will be extracted. The diameter and the color of the dots varied with the amplitude and frequency of the music, and the pictures thus they were painted with beautiful colors. (see Sound to light)
COMBINE ART & SCIENCE!As Wilde conveyed in his story, the barrier and combination of art and science still remain worth discussing, so we explored in depth the relationship of art and science in the Human Practices section. One prime barrier of outstanding art and science is the stereotype of defining art as being too selfish and far from the public and science as only about reality with no emotion. We surveyed university students from science and art backgrounds, and to our surprise we found the unconventional idea that the integration of art and science has been well-accepted among the younger generation.
Encouraged by our survey , we interviewed many university professors who are experienced in popularization of both art and science, and also communicated with artists in the AS Research Center. During this investigative interview process we gradually got motivated and had a clearer idea of our story to explore the junction of art and science.(see HP)
More than a hundred years ago, in Wilde’s story, the rose was thrown into the gutter. But today, we UCAS-China iGEMers have picked the rose back up from the gutter, to offer everyone the chance to create their own rose. Inspired by idealism and stirred by imagination, facilitated by scientific gene circuits we develop a practical kit expecting that our work will inaugurate a new era of art and science further inspiring young scientists for future generations.
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] https://2010.igem.org/Team:KIT-Kyoto
[5] Cong L, Zhang F. Genome Engineering Using CRISPR-Cas9 System[J]. Methods in Molecular Biology, 2015, 1239(11):197.
[6] 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.
[7] https://2014.igem.org/Team:Paris_Bettencourt
Design Design 2
We introduced the RGB system[1] to stain our rose using light. The RGB system mainly consists of four modules: a ‘sensor array’, ‘circuits’, a ‘resource allocator’ and ‘actuators’.
The ‘sensor array’ combines 3 light sensors, Cph8*(BBa_K2598006), YF1 (BBa_K2598009) and CcasR (BBa_K2598005), 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. Cph8* and CcaSR are based on phytochromes in the phycocyanobilin chromophores, which are produced by pcyA and ho1(BBa_K2598018)[3], while YF1 is based on Flavin mononucleotide(FMN) chronmophore.
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 repressor CI turns off the promoter(P) of K1F, and red light can release the inhibition effect and induce the P promoter. Respectively, the repressor PhlF turns off the promoter(PPhlF) of T3, and blue light can induce the PPhlF. After the process of circuits, red-, green- and blue-light signals can respectively induce the P, PcpcG2-172 and PPhlF promoters.
Design 3
The ‘resource allocator’ which connects the ‘circuits’ and ‘actuators’, is based on a split-RNA polymerase(RNAP) system[2]. In the system, a non-active ‘core’ fragment is constitutively expressed by promoter J23105. The sigma fragments(K1F, CGG, T3) regulated by P, PcpcG2-172 and PPhlF can conjugate with the core fragment to form full-functional RNA polymerases, and the RNA polymerases are then directed to three promoters and activated the expression of actuators respectively. It is worth mentioning that the sigma fragments are chosen that exhibited the least cross-talk. Furthermore, insulators (BydvJ, BBa_K2598010, and RiboJ[4], BBa_K2598014) were added to reduce the crosstalk among the three sigma fragments.
The actuators we chose were three kinds of color protein: fluorescent protein, chromoprotein and enzyme which can produce colorful products. The fluorescent proteins mRFP(BBa_K2598065), GFP(BBa_K2598063), BFP(BBa_K2598064) were chosen to test our system and achieve our final results because fluorescent reporters can be measured easily by ELIASA (microplate reader) and flow cytometry and the expression period is much shorter than those of chromoprotein and enzyme. What’s more, we use chormoproteins amilGFP (BBa_K2598055), amilCP (BBa_K2598057) and eforred (BBa_K2598056) to prove our concept. We also constructed a plasmid containing lacZ(BBa_K2598025), bFMO(BBa_K2598029) and gusA(BBa_K2598032), which are enzymes and can generate colorful pigments on the plates. The enzyme LacZ, bFMO and gusA can react with X-gal, tryptophan and Rose-gluc, respectively. The products of these enzymes can form insoluble precipitates and color the plates. We also used enzymes to test our system under a more complexed situation, to prove our concepts.
Design 4 Figure 3. The compositions of four plasmids we constructed.
Six plasmids composed the whole system, pJFR1 (BBa_K2598049), pJFR2 (BBa_K2598050), pJFR3(BBa_K2598051), pJFR4(BBa_K2598053), pJFR5(BBa_K2598052) and pJFR6(BBa_K2598061). The ‘sensor array’ module and the core fragment was integrated into pJFR1. The ‘circuits’ and ‘resource allocator’ were integrated into pJFR2 and pJFR3. And the ‘actuators’ containing fluorescence proteins, chormoproteins and enzymes were integrated into pJFR4, pJFR5 and pJFR6, respectively. We transformed four plasmids into our E.coli and finished our artwork.(For more information, see Parts)
Mixing Colors in Cells!Although more fluorescent proteins and chromoproteins are edited to generate more and more colors, the number of colors produced by organisms is still limited by the number of the kinds of proteins. Once a new color is needed, researchers have to modify the chromophores of the proteins, which takes much time and effort.
So how do we create more colors, to make our rose more colorful in a reasonable and convenient way?Here, we put forward a new concept—mixing color in bacterial cells! Unlike the mix of different bacterial cells which produce different colors as the previous iGEM teams have done[5], we used tandem expression and RGB system to control the ratios of the expression of different colors in bacterial cells, to achieve mixing color in bacterial cells, and stain our roses with more bright colors.
Mixing Color by Tandem Expression! Figure 4. The color spectrum built from chromoproteins and their tandem expression products.
To prove our concept, we firstly use the tandem expression of chromoproteins. By putting two chromoprotein RBSs and genes under one promoter, we constructed six plasmids(BBa_K2598043, BBa_K2598044, BBa_K2598045, BBa_K2598046, BBa_K2598047, BBa_K2598048) to tandem express eforred, amilCP, amilGFP and fwYellow chromoproteins.
As shown in the figure 4, we built a color spectrum using chromoproteins and their tandem expression products, which can show orange, pink, yellow and green colors. Furthermore, we can see that the color of the tandem expression products, is always between the colors of the two chromoproteins in the spectrum. That is to say, by applying the physical principles, we can easily mix the color we want using tandem expression of chromoproteins.
Mixing 2 Figure 5. The position of the color of GFP and the mixed color by the tandem expression of amilCP and amilGFP in the spectrum.
What’s more, using the tandem expression of amilCP and amilGFP, which are blue and yellow respectively, we mixed blue and yellow together to create green color, which rarely exist in colors of chromoproteins in iGEM registry. We use color picker and Photoshop to analyze and compare the color we got and that of amilGFP. The RGB value of our color is 184-209-108, while that of amilGFP is 178-191-138. That shows our color from the tandem expression is brighter and greener. Following the physical principles of mixing color and tandem expression rather than struggling to edit the molecular structure of the proteins, we provided a new approach to getting a new color with less cost of time and effort, which will largely enrich the part registry and generate more colors for scientific research and art creation.
Mixing Color by Applying RGB system!We performed simple verification of our concept using tandem expression and got well-fitted results, but the bacteria tandem express several chromoproteins can only have one color at a time, so we tried to use more convenient methods, for example light, to control and change the color of the bacteria. We decided to use the RGB system, which was introduced above, to achieve light-controlled color mix in bacteria, because light sensors on the cell membrane can response differently to the lights with different wavelengths, thus producing different outputs accordingly. By changing the wavelengths of the lights, we can control the response of red-, green- and blue-light sensors and expression of mRFP, GFP and BFP. And for precise quantitative data, we chose fluorescent proteins (GFP, BFP, mRFP) as our actuators to mix the colors. What’s more, to avoid the influence of leakage of the fluorescent proteins and get more colors, we also constructed 3 plasmids which only contained 2 output fluorescent protein genes (GFP & mRFP, BFP & mRFP, GFP & BFP), and transformed into the E.coli with pJFR1, pJFR2, pJFR3.
We also tried to induce the expression of fluorescent proteins using the input light which was mixed by lights from different wavelengths. We used mixed light to induce the system because the light from the projectors was mixed light of red-, green-, and blue-light, which we would use in our final hardware. (More information, see HARDWARE)
Mixing II Figure 6. The process of overlapping the fluorescent pictures, filtering the colors and get the results.
We chose lights of 13 wavelengths from 395 nm to 660 nm, to test our system and produce colors. We firstly use light to illuminate our bacteria for hours on the plate, and then use fluorescence camera to excite the red, green and blue fluorescence and capture the photos. Then using imageJ and Photoshop, we can overlap the three pictures, filter the colors and get our results of mixing colors(Figure 6).
Mixing III Figure 7.The color spectrum built from fluorescent color mixing.
The figure 7 demonstrated the results of our fluorescent color mixing. Using color picker to pick out the mixed colors from our pictures, we built a even wider spectrum with more colors, with which we created a colorful rose as shown in the figure 8. This result further proved that our system worked and our concept that we can use convenient method—light control—to achieve the mixing and changing the colors of the bacteria was valid.
Mixing IV Figure 9. The curves showing the relationship between the R-G-B value and the wavelengths of light
To semi-quantitatively analyze the fluorescent plate results, we extracted the RGB value of the pictures and got curves showing the relationship between the R value, G value and B value and the wavelengths of the light. From the figure 9 we could see the trend and the peaks of the curves, that the B value reached the top at the wavelength of 490nm, the G value at 565nm, and the R value at 620nm. The R, G, B value could roughly show the expression of red, green and blue fluorescent proteins and their fluorescence intensity, greatly fitted with the literature data that the blue-light sensor was induced best at 470nm, green-light sensor at 532nm, and red-light sensor at 650nm. These curves further proved the function of our system.
Mixing V Figure 10. 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.
For more precise control and prediction of the colors we could generate, we needed to know how the three light sensors responded to lights with different wavelengths and intensity and how the fluorescence changed with time. We firstly use flow cytometry[6] to measure the spectral response of the RGB system. From the figure 10 we could see 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. The figure demonstrated the variation of fluorescence intensity where cells were most concentrated with the wavelengths of light. The red- and blue- light data were consistent with our prediction. However, the green-light data was not so satisfying, which was probably because without NOT gate, the green-light circuit had leakage and produced fluorescent proteins even not induced by light. What’s more, the different culture and light-inducing conditions of bacteria on the plates and 96-well plates might also cause the slight inconsistency.(For more details of our optimization, please see HARDWARE)
Mixing VI Figure 12. The curves show the relationship between the input light and fluorescent intensity.
From the flow cytometry results, we knew the correspondence between color and wavelengths of light. We then controlled the intensity of the input light to see the change of the fluorescent intensity over time. The figure 12 demonstrated that the stronger the input light was, the less fluorescence would be. The phenomenon was probably because that under relatively low light, the light sensor could be induced and under relatively strong light, the light can aggravate the burden of the bacteria and thus harmed the growth of the bacteria and reduced the expression of fluorescent proteins. We modelled this result for further explanation.(For more information, please see MODEL)
After using fluorescent proteins, we used the plasmid pJFR5 (BBa_K2598052), which contained enzyme genes lacZ (BBa_K2598025), bFMO (BBa_K2598029) and gusA (BBa_K2598032), to created more colors using enzymes. The enzymes could generate pigments on the plates and the results of mixing color was easier to observe, which could provide a new material for artists to create artworks. From the figure 13, we could see the different colors generated by enzymes and our system also worked well. The colors were a little dark, and we thought that the phenomenon might resulted from the high concentrations of the substances and long illumination period, which could be adjusted in the future.
Mixing VII Figure 13. The results of mixing color using enzyme.
By controlling the wavelength and intensity of the input light and the time of exposure, we could predict and control all kinds of colors our bacteria would produce. We did not have to edit the proteins to get a new color, and all we needed to do was to change the input light. Our concept of mixing color on the bacteria cells were proved to be reasonable and provided a convenient way for scientists and artists to create new colors and artworks. Applying the RGB system to our E.coli, now we could now stain our rose with various beautiful colors using colorful lights!
Reference:
[1] 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.
[2] 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.
[3] Gambetta G A, Lagarias J C. Genetic engineering of phytochrome biosynthesis in bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(19):10566-10571.
[4] Lou C, Stanton B, Chen Y J, et al. Ribozyme-based insulator parts buffer synthetic circuits from genetic context.[J]. Nature Biotechnology, 2012, 30(11):1137-1142.
[5] https://2010.igem.org/Team:KIT-Kyoto
[6] Davey H M, Kell D B. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses[J]. Microbiol Rev, 1996, 60(4):641-696.
Gene knock-out using CRISPER-Cas9 Figure 1. The tnaA and L-tryptophan degradation resulted in the unpleasant odor in E. coli
Our roses were colorful now, but only fragrance that makes them vivid, appealing and with the soul as a real rose. So there came our second part, light to odor, making our roses more perfect and real. We first tried to use CRISPR/Cas9[1] gene-editing system to knock out the original gene of E. coli producing smell to prevent E. coli from giving off a nasty and unpleasant smell. Then based on the RGB system[2], we achieved the control of multiple odors with light.
The inherent unpleasant odor of E. coli comes from indole produced naturally in the cells’ metabolic process. In the L-tryptophan degradation pathway, Tryptophanase, encoded by tnaA gene, degraded L-tryptophan of indole, which produces the odor in high concentration.[3]
Knock out Figure 2.The procedure and the mechanism of CRISPER-Cas9
Therefore, using CRISPR/Cas9 system, we tried to knock out the tnaA gene. First, we transformed a pCas9 plasmid into the host cell. Then selecting the positive clone, we used electroporation to transform plasmid TargetF (Figure 4) with a pair of specific sgRNA, and a piece of homologous DNA (Figure 5) which consists of the two gene segments located at the either side of tnaA. Guiding by the sgRNA, Cas9, a nuclease, cut the tnaA segment, leaving a leakage between the two homologous DNA. The cell tended to keep the chromosome integrity, so there was the probability to repair the leakage with the piece of homologous DNA we transformed before, because they have two paired sequences on either side. Also, we gave higher-fold of homologous DNA to increase this probability. After the whole process, we needed to cure the exogenous DNA we introduced.
这里还要加一个表格。 Results Figure 4. The PCR results of pTarget
Figure 5. The PCR results of Homologous sequence 1, 2, 1+2
Fragrance introducing
Then we devoted our effort to introducing diversified odor to make our roses not only vivid ones with traditional flower fragrance, but unique ones with more kinds of odor like lemon and rain. So we improved part of the iGEM project of Paris_Bettencourt in 2014 [4]by putting their parts into our RGB system and under the control of light. What’s more, we also made some adjustments to the concentrations of substrates provided by Paris_Bettencourt in 2014[5]. As tested in the LIGHT TO COLOR(链接), the RGB system functioned well, no matter whether the actuators was fluorescent proteins or enzymes. But for further validation, we used HPLC to detect the products of our actuators.
We constructed a plasmid (BBa_K2598062) containing lims1 (BBa_K2598060), gds (BBa_K2598058), bmst1 (BBa_K2598059) as actuators, which produced the smell of lemon, rain and flower respectively. The function of the system and the actuators was shown in Table 1. Then we transformed the plasmids of RGB system into E.coli, cultured the bacteria by 96-well plates and induced the expression of odor enzymes by light. (More details, please see Hardware)
SomewhatWe constructed a plasmid (BBa_K2598062) containing lims1 (BBa_K2598060), gds (BBa_K2598058), bmst1 (BBa_K2598059) as actuators, which produced the smell of lemon, rain and flower respectively. The function of the system and the actuators was shown in Table 1. Then we transformed the plasmids of RGB system into E.coli, cultured the bacteria by 96-well plates and induced the expression of odor enzymes by light. (More details, please see Hardware)
HPLC was applied to detect our products in the fluid LB medium. However, we faced great difficulties because rare previous literature had reported the mobile phase, the temperature, and even the parameter settings of the three molecules, especially geosmin and methyl benzoate. So we chose acetonitrile-water, which is the most common mobile phase, to roughly separate the substances in the LB medium and 200nm as the detection wavelength, which was commonly used to detect Limonene[6].
HPLC results Figure 6. The HPLC results of the standard samples of Limonene, Methyl benzoate and Geosmin.
The HPLC results of standard samples were shown in Figure 6, while that of our samples induced by Blue light was shown in figure 7. The retention times of Limonene, Methyl benzoate and Geosmin were 8.770min, 3.764min, 2.835min, respectively. The peaks were found in our HPLC results of our samples, but the peaks were not divided and further analysis (for example MS) were required to determine the substances which produced these peaks. What’s more, 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 details of the function of our system, see LIGHT TO COLOR)
Reference Figure 7. The HPLC result of the sample induced by blue light. Peaks at 2.703min, and 3.743min were found, which were closed to the peaks of methyl benzoate and geosmin. But further analysis (for example mass spectrometry, MS) were also required to determine the substances which produced these peaks.
Reference:
[1] Cong L, Zhang F. Genome Engineering Using CRISPR-Cas9 System[J]. Methods in Molecular Biology, 2015, 1239(11):197.
[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] 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.
[4] https://2014.igem.org/Team:Paris_Bettencourt
[5] http://parts.igem.org/Part:BBa_K1403006
[6] Ruilun Z., Qing Z., Shiai XU. Determination of Limonene in Spearmint Oil by HPLC method[J]. Journal of Anhui Agri, 2012, 40(2):743-744.
Sound To Light Figure 1.The schema of our software Orpheus.
In Oscar Wilde’s story, it was the singing of the Nightingale that brought a unique soul to the rose. Correspondingly, in our project, we needed different sounds to bring distinctive souls to our roses. Therefore, Orpheus was born!
Our software was named after the god of music in Greek mythology.[1] When he played his harp the world would sway to the music. Sadly, he lost his beloved wife and failed to bring her back from the underworld. After his death, Orpheus was buried at Leibethra below Mount Olympus, where the nightingales sang over his grave. We named our software Orpheus in memory of this god of music. We hope the beautiful songs and touching roses created by our software can bring comfort and sincere wishes everyone.
Orpheus is an online software which can convert any sound into corresponding color spots and paint them on an inputted image.
Process Figure 2. The operation of our software Orpheus.
First of all, users can choose an image file and an audio file stored in their computer. Then Orpheus will graying the inputted image and carefully decodes the inputted audio into wave shapes. After that a series of spots will be generated with corresponding hue and brightness on the image. For hue, Orpheus first decodes sound into wave shapes and analyzes its low, medium and high volume, then generates RGB values with our delicate mapping function. For brightness, Orpheus converts source photos into grayscale images which is in proportion to brightness. The random dropping process, just as the nightingale slowly colors the rose as it warbles in the story, each drop will color some area by the aforementioned method accompanied by the inputted music and finally a beautiful picture is produced.
这里还要加三个视频的网页 Something good 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.
With Orpheus，everyone can put a unique soul into his rose. Combined with our hardware, we can finally create a rose forest, permeated with beauty and romance, for all human-beings! Interested? Why not try it now? Don’t forget to upload your final image to our online album and share it to everyone! Software link: 这里加一个链接
Something better Figure 3. We combined tons of pictures from the software and creatively made a composite image of our banner.
Using Orpheus, we everyone could ensoul the roses with our own voice, our music, and even the natural sounds. The interaction designed here also resulted from our human practices, which shew us that the acceptance of the combination of art and science among young generation was high and what we needed was to provide them a practical chance to be involved in the art and science. So we created Orpheus, wishing that everyone would enjoy the art-and-experience experience when they brought roses unique souls.(More details, please see Human Practices)
Reference
[1]https://simple.wikipedia.org/wiki/Orpheus
The RGB system in our project Figure 1. The RGB system composed of four subsystems(sensor array, circuits, resource allocator and actuators) is shown.
In our project we introduced the RGB system[1] to stain and to scent our rose using light. The RGB system mainly consists of four modules: a ‘sensor array’, ‘circuits’, a ‘resource allocator’ and ‘actuators’. The ‘sensor array’ combines 3 light sensors, Cph8*(BBa_K2598006), YF1 (BBa_K2598009) and CcasR (BBa_K2598005), 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. Cph8* and CcaSR are based on phytochromes in the phycocyanobilin chromophores, which are produced by pcyA and ho1(BBa_K2598018)[3], while YF1 is based on Flavin mononucleotide(FMN) chronmophore.
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 repressor CI turns off the promoter(P) of K1F, and red light can release the inhibition effect and induce the P promoter. Respectively, the repressor PhlF turns off the promoter(PPhlF) of T3, and blue light can induce the PPhlF. After the process of circuits, red-, green- and blue-light signals can respectively induce the P, PcpcG2-172 and PPhlF promoters.
Genes Figure 2.The RGB system is encoded on 4 plasmids. The genes and genetic parts are shown.
The ‘resource allocator’ which connects the ‘circuits’ and ‘actuators’, is based on a split-RNA polymerase(RNAP) system[2]. In the system, a non-active ‘core’ fragment is constitutively expressed by promoter J23105. The sigma fragments(K1F, CGG, T3) regulated by P, PcpcG2-172 and PPhlF can conjugate with the core fragment to form full-functional RNA polymerases, and the RNA polymerases are then directed to three promoters and activated the expression of actuators respectively. It is worth mentioning that the sigma fragments are chosen that exhibited the least cross-talk. Furthermore, insulators (BydvJ, BBa_K2598010, and RiboJ[4], BBa_K2598014) were added to reduce the crosstalk among the three sigma fragments.
The “actuator” module implement the biological functions that are the outputs of the RGB system. There are three output genes responding to three light sensors activated by different wavelength of light. In order to implement customized outcome, theoretically, you can change them into any genes you want. The actuators we chose here were three kinds of color protein: fluorescent protein, chromoprotein and enzyme which can produce colorful products. The fluorescent proteins mRFP(BBa_K2598065), GFP(BBa_K2598063), BFP(BBa_K2598064) were chosen to test our system and achieve our final results because fluorescent reporters can be measured easily by ELIASA (microplate reader) and flow cytometry and the expression period is much shorter than those of chromoprotein and enzyme. What’s more, We use chormoproteins amilGFP (BBa_K2598055), amilCP (BBa_K2598057) and eforred (BBa_K2598056) to prove our concept. We also constructed a plasmid containing lacZ(BBa_K2598025), bFMO(BBa_K2598029) and gusA(BBa_K2598032), which are enzymes and can generate colorful pigments on the plates. The enzyme LacZ, bFMO and gusA can react with X-gal, tryptophan and Rose-gluc, respectively. The products of these enzymes can form insoluble precipitates and color the plates. We also used enzymes to test our system under a more complexed situation, to prove our concepts.
Part Compositions Figure 3.The compositions of four plasmids we constructed, the actuators in pJFR4 could be replaced by chromoproteins and enzymes.
Six plasmids compose the whole system, pJFR1 (BBa_K2598049), pJFR2 (BBa_K2598050), pJFR3(BBa_K2598051), pJFR4(BBa_K2598053), pJFR5(BBa_K2598052) and pJFR6(BBa_K2598061). The ‘sensor array’ module and the core fragment was integrated into pJFR1. The ‘circuits’ and ‘resource allocator’ were integrated into pJFR2 and pJFR3. And the ‘actuators’ containing fluorescence proteins, chormoproteins and enzymes were integrated into pJFR4, pJFR5 and pJFR6, respectively. We transformed four plasmids into our E.coli and finished our artwork.(For more information, see Parts)
The concept: Mixing colors in bacteria cellsAlthough more and more fluorescent proteins and chromoproteins are edited to generate more and more colors, the number of colors produced by organisms is still limited by the number of the kinds of proteins. Once a new color is needed, researchers have to modify the chromophores of the proteins, which takes much time and effort.
So how do we create more colors, to make our rose more colorful in a reasonable and convenient way? Here, we put forward a new concept—mixing color into bacterial cells! Unlike the mix of different bacterial cells which produce different colors as the previous iGEM teams have done, we used tandem expression and RGB system to control the ratios of the expression of different colors in bacterial cells, to achieve mixing color in bacterial cells, and stain our roses with more bright colors.
Primary proof of our concept: the tandem expression
Figure 4. The color spectrum built from chromoproteins and their tandem expression products.
As shown in the figure 4, we built a color spectrum using chromoproteins and their tandem expression products, which can show orange, pink, yellow and green colors. Furthermore, we can see that the color of the tandem expression products, is always between the colors of the two chromoproteins in the spectrum. That is to say, by applying the physical principles, we can easily mix the color we want using tandem expression of chromoproteins.
Green Color Figure 5. The position of the color of GFP and the mixed color by the tandem expression of amilCP and amilGFP in the spectrum
What’s more, using the tandem expression of amilCP and amilCP, which are blue and yellow respectively, we mixed blue and yellow together to create green color, which rarely exist in colors of chromoproteins in iGEM registry. We use color picker and Photoshop to analyze and compare the color we got and that of amilGFP. The RGB value of our color is 184-209-108, while that of amilGFP is 178-191-138. That shows our color from the tandem expression is brighter and greener. Following the physical principles of mixing color and tandem expression rather than struggling to edit the molecular structure of the proteins, we provided a new approach to getting a new color with less cost of time and effort, which will largely enrich the part registry and generate more colors for scientific research and art creation.
Function of RGB system & Further Proof of conceptWe performed simple verification of our concept using tandem expression and got well-fitted results, but the bacteria tandem express several chromoproteins can only have one color at a time, so we tried to use more convenient methods, for example light, to control and change the color of the bacteria. We decided to use the RGB system, which was introduced above, to achieve light-controlled color mix in bacteria, because light sensors on the cell membrane can response differently to the lights with different wavelengths, thus producing different outputs accordingly. By changing the wavelengths of the lights, we can control the response of red-, green- and blue-light sensors and expression of mRFP, GFP and BFP. And for precise quantitative data, we chose fluorescent proteins (GFP, BFP, mRFP) as our actuators to mix the colors. What’s more, to avoid the influence of leakage of the fluorescent proteins and get more colors, we also constructed 3 plasmids which only contained 2 output fluorescent protein genes (GFP & mRFP, BFP & mRFP, GFP & BFP), and transformed into the E.coli with pJFR1, pJFR2, pJFR3.
Noname Figure 6. The process of overlapping the fluorescent pictures, filtering the colors and get the results.
We chose lights of 13 wavelengths from 395 nm to 660 nm, to test our system and produce colors. We firstly use light to illuminate our bacteria for hours on the plate, and then use fluorescence camera to excite the red, green and blue fluorescence and capture the photos. Then using imageJ and Photoshop, we can overlap the three pictures, filter the colors and get our results of mixing colors(Figure 6).
Noname 2 Figure 7. The color spectrum built from fluorescent color mixing.
Figure 8. The rose we created by using the colors we mixed.
The figure 7 demonstrated the results of our fluorescent color mixing. Using color picker to pick out the mixed colors from our pictures, we built a even wider spectrum with more colors, with which we created a colorful rose as shown in the figure 8. This result further proved that our system really worked and our concept that we can use convenient method—light control—to achieve the mixing and changing the colors of the bacteria was true.
Noname 3 Figure 9.The curves showing the relationship between the R-G-B value and the wavelengths of light
To semi-quantitatively analyze the fluorescent plate results, we extracted the RGB value of the pictures and got curves showing the relationship between the R value, G value and B value and the wavelength of the light. From the figure 9 we could see the trend and the peaks of the curves, that the B value reached the top at the wavelength of 490nm, the G value at 565nm, and the R value at 620nm. The R, G, B value could roughly show the expression of red, green and blue fluorescent proteins and their fluorescence intensity, greatly fitted with the literature data that the blue-light sensor was induced best at 470nm, green-light sensor at 532nm, and red-light sensor at 650nm. These curves further proved the function of our system.