Team:Lethbridge HS/Design

Design

Environmental Impact

Constructs

During the pigment selection process we focused on pigments that would be safe for the environment and safe to humans (i.e. non-pathogenic) as well as the yield of pigments in the bacteria, we also made sure that we had some papers backing our research in that we found some that had previously produced the pigments in our chosen bacteria escherichia. Coli. For our four pigments we have selected Indigoidine for cyan, Anthocyanin for magenta, Zeaxanthin for yellow and Melanin for black. All of our constructs will be using a T7 promoter and an Escherichia coli Ribosomal Binding Site, as well as a T7 Terminator. The constructs will be in the plasmid pSB1C3, for our expression in E. coli strain bl21(DE3). We also used the plasmid DH5Alpha for the production of more of our construct DNA before we put it into bl21(DE3) for expression.

T7 Promoter system

The T7 system is a system by which IPTG can be introduced into cells for the transcription and translation of genes. T7 is an E. coli virus, which the gene for the T7 RNA polymerase has been taken from. The E. coli strain BL21(DE3) contains a construct within its DNA that has an inducible promoter at the front of it. The promoter is lactose inducible but we will be using IPTG to induce the promoter as it has a similar structure (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