Team:Osaka/COLOR

COLOR

Overview We have tried to develop new art tools in addition to creating our own bio-art. New tools will open new possibilities for art. We want to provide outstanding 'painting' tools. So far, we have incorporated fluorescent proteins and organic pigments into our art tool ‘colrcoli’. We aim to create as diverse and extensive a palette of art and coloring tools as possible.

Object Color is a very important aspect of art. The colors used for 'colacoli' consist of fluorescent as well as non-fluorescent pigment colors. Our palette of fluorescent colors include red, green, cyan, yellow, and orange, made possible by incorporating RFP, GFP, CFP, YFP, and mOrange fluorescent proteins respectively. We actually wanted to have even more colors, but the parts coding for fluorescent proteins supplied in the 2009 iGEM DNA distributiont were of limited variety. So we adopted a different method for obtaining new colors, that is by non-fluorescent organic pigments. Pigment colors are red, orange, yellow, brown, black, and purple. Red, orange, and yellow colors can be provided by carotenoids, brown and black by melanin and purple by violacein. Fluorescence We assembled genetic circuits for fluorescence colors. We used two patterns promoters, p(Lac) or p(Tet), for each fluorescent protein coding device. Carotenoid Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants. Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP).  IPP and DMAPP are formed in mevalonate pathway or nonmevalonate pathway. Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. And nonmevalone pathway is to produce isoprenoids in plants and apicomplexan protozoa. We succeeded in assembling the device coding for carotene (orange color). But the color of the cell colonies was quite weak, and giving a cream rather than orange color.  Melanin Melanin is a pigment that is made in the human body. There are brown-black melanin pigments and orange-red melanin pigments. For example, our hair contains melanin as a major pigment. So the quantity of melanin makes natural hair color. Biosynthesis of melanin pigments starts from tyrosine which is one of the amino acids. Tyrosine is changed to dopa and dopaquinone by the action of tyrosinase. Finally products become brown-black melanin. 

Violacein Violacein is a purple pigment. Bacteria in deep seawater produce this pigment. Violacein is claimed to have antibacterial and anti-tumor properties. Biosynthesis of violacein pigments starts from tryptophan. As a substrate with tryptophan, to produse violacein needs enzyme vioA - vioE.  We tried to find DNA parts coding for the melanin and violacein pigments but couldn't. So we were unable to produce the colors brown, black, and purple.

Collaboration with sensor We assembled the genetic circuits that turn on their colors upon activation by a sensor switch. When a receiver of a signaling system gets a signal, a downstream gene that produces a certain color is activated. In the absence of a signal, they will have no color. However we were unable to make a perfect on/off system. Even without a signal, weak 'leaky' transcription occurs causing color to be faintly expressed. However the sensor does make a difference in the level of expression. So we made use of the differences to check the functionality of the sensors, as shown in these circuits below. p(TetR)+RBS+Receiver(see SIGNAL.)+tt+P(Receiver+signal)+RBS+color+tt 

Future Work

Color gradation We designed a the genetic circuit which will gradually change cell color. This gene circuit indicates bellow. This is an application of our signal circuit. Signal system includes 2 distinct groups of parts: 'Senders' and 'Receivers'. The specific promoter in the receiver cells is activated by receiving the AHL signal from the sender cells. (see SIGNAL.)

On this device, there is a gene coding for an initial color, AHL receiver coding region, a gene coding for a different color and a CI lambda protein coding region. The regions coding for secondary color and CI lambda protein are situated downstream of AHL-activated promoter. The CI lambda protein is the repressor of the CI promoter which regulates production of the initial color. Therefore, when the signal is received, the cell gradually starts expression of the new color protein and stops expression of initial color protein. In addition, the same cell will also code for autoinducer producing proteins for another AHL signaling system, which will let it act as a sender to other cells. The planned circuit is as shown below.



Below movie indicates ideal results. The simulation movie includes an unknown parameter because of no experiment. 

