Environmental Impact

Constructs

We propose to produce biological pigments that can be used in ink at a smaller environmental impact than current sources. We have identified pigments that can be produced in Escherichia coli that correspond to the four main colors in ink cartridges: black, cyan, magenta, and yellow. They are melanin, indigoidine, anthocyanin, and zeaxanthin, respectively. We have decided to produce these pigments because there has been success in producing these pigments in the past ( Misawa et al., 1990, Cabrera-Valladares et al., 2006, Lagunas-Muñoz et al., 2006,Yan et al., 2008, Lim et al., 2015, Xu et al., 2015). Using synthetic biology techniques, genes corresponding to pigment biosynthetic pathways from a variety of organisms will be engineered for expression in E. coli. Pigment production will be followed by purification to separate the pigments from the bacteria, which will then allow the produced pigments to be incorporated into ink.

Standard Genetic Construct for Pigment Production

Each of the four pigment production constructs is designed in the same way. Each construct consists of a T7 promoter, the E. coli RBS B0034 (with the exception of the zeaxanthin construct, which uses Pantoea ananatis native RBSs), the coding sequences for pigment biosynthetic genes, and the double terminator B0015.

Use of a T7 promoter allows us to utilize the induction system of the E. coli strain BL21(DE3). In this strain, T7 RNA polymerase (RNAP) is expressed by the lacUV5 promoter, which can be induced by the addition of IPTG to the culture. T7 RNAP will then transcribe the pigment biosynthesis constructs(Fig 1.).

Fig 1. a comparison of the two molecules IPTG and Lactose

The T7 ribosomal binding site will then attach to the T promoters on our constructs and perform translation on the DNA sequence making an mRNA strand. This system allows us to have the greatest expression of our gene as the T7 promoter can only act on our construct and thus it will undergo transcription more frequently than if it required an E. coli RBS.

Indigoidine

We used the genes indB and indC from the organism Pantoea ananatis in the plasmid pSB1C3. We chose indigoidine because it has been expressed in E. coli, as well as our research showed that it was not harmful to humans or the environment. These genes converted the initial molecule l-Glutamine into Indigoidine (Fig 2). There are multiple organisms that produce Indigoidine and we chose our gene from P. ananatis because they have been used in E. coli as well as we will be adding it to the iGEM registry.

Fig 2. The pathway from l-Glutamine to Indigoidine and its associated genes.

Anthocyanin

The Magenta pigment we decided on was Anthocyanin. The genes we used are; dfr from the organism Anthurium andraeanum, F3h and ans from Malus domestica, and 3gt from Petunia hybrid. We chose these genes from these organisms as they have been expressed in E. coli as well as the anthocyanin has been proved non-harmful to humans and the environment. These genes converted the initial molecule Eriodictyol into our final molecule Anthocyanin (Fig 3). Team Darmstadt 2014 made the molecule pelargonidin for their use in a bio solar panel.

Fig 3. The anthocyanin synthesis pathway, from our initial molecule Eriodictyol into our final molecule Anthocyanin.

Zeaxanthin

The yellow pigment we used is Zeaxanthin, which is a part of the Carotenoid synthesis pathway. The genes we used were CrtY and CrtZ from the organism P. ananatis. These two genes create enzymes that turned our initial molecule Lycopene into the molecule Zeaxanthin (Fig 4). We will supplementing our media with Lycopene because it allows us to skip three genes in the pathway and improve our chances of success. These genes were selected as they have been produced in E. coli as well as the safe final product.

Fig 4. The carotenoid synthesis pathway and our initial and final molecules. As well as the genes that we skipped by adding Lycopene to our media.

Melanin

We chose Melanin as our Key color, the black. It was selected because it has easily been expressed in E. coli as well as it is safe for humans and the environment. The gene we used was elA from the organism Rhizobium etli because it has been produced before, it converts L-Tyrosine into Dopaquinone which then undergoes a non-enzymatic natural chain reaction to form Melanin (Fig 5). This part is in the registry as BBa_K274001, originally created by team Cambridge 2009 and further characterized by Concordia 2016 since.

Fig 5. The pathway from L-tyrosine to Melanin with the use of the melA gene.

References

Xu, F., Gage, D., and Zhan, J. (2015) Efficient production of indigoidine in Escherichia coli. Journal of Industrial Microbiology & Biotechnology. 42, 1149–1155

Yu,D., Xu, F., Valiente, J., Wang, S., and Zhan, J. (2012) An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue. Journal of Industrial Microbiology & Biotechnology. 40, 159–168

Cude, W. N., Mooney, J., Tavanaei, A. A., Hadden, M. K., Frank, A. M., Gulvik, C. A., May, A. L., and Buchan, A. (2012) Production of the Antimicrobial Secondary Metabolite Indigoidine Contributes to Competitive Surface Colonization by the Marine Roseobacter Phaeobacter sp. Strain Y4I. Applied and Environmental Microbiology. 78, 4771–4780

Brachmann, Alexander O., Ferdinand Kirchner, Carsten Kegler, Sebastian C. Kinski, Imke Schmitt, and Helge B. Bode. "Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens." Journal of Biotechnology 157.1 (2012): 96-99. Web.

Yan, Y., Li, Z., and Koffas, M. A. (2008) High-yield anthocyanin biosynthesis in engineered Escherichia coli. Biotechnology and Bioengineering. 100, 126–140

Lim, C. G., Wong, L., Bhan, N., Dvora, H., Xu, P., Venkiteswaran, S., and Koffas, M. A. G. (2015) Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin. Applied and Environmental Microbiology. 81, 6276–6284

Sedkova, N., Tao, L., Rouviere, P. E., and Cheng, Q. (2005) Diversity of Carotenoid Synthesis Gene Clusters from Environmental Enterobacteriaceae Strains. Applied and Environmental Microbiology. 71, 8141–8146

Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K., and Harashima, K. (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. Journal of Bacteriology. 172, 6704–6712

Lagunas-Muñoz, V., Cabrera-Valladares, N., Bolívar, F., Gosset, G., and Martínez, A. (2006) Optimum melanin production using recombinant Escherichia coli. Journal of Applied Microbiology. 101, 1002–1008

Cabrera-Valladares, N., Martínez, A., Piñero, S., Lagunas-Muñoz, V. H., Tinoco, R., Anda, R. D., Vázquez-Duhalt, R., Bolívar, F., and Gosset, G. (2006) Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase. Enzyme and Microbial Technology. 38, 772–779