Prior to working directly in fish,we needed to set out our experiments in a relatively safe and predictable chassis. The simplicity of a bacterial molecular and regulator system, their ability to divide exponentially, and carry out complex tasks has provided Synthetic Biologists an ability to genetically engineer their genome in order to find eco-friendly solutions. E.coli serves as a valuable chassis for such systems which have real world implications. (7)
Our Test-phase Detection Systems will be inserted inside an E-coli DH5α cell, which will be heavy-metal specific and shall work only in the presence of contaminants in the water resource.
The system will consist of metal inducible promoters which are sensitive to the exposure of heavy-metals. These heavy-metal inducible promoters of four of our devices are regulated by regulators; activators and repressors which activate the promoter only in the presence of the specific heavy metal. These promoters are placed under control of a constitutive promoter. The activated promoters initiate the translation of reporter genes, ultimately expressing chromoproteins. This is the basic concept of our biosensor which will allow rapid and simultaneous detection of six heavy metals in water.
We utilized arsenic inducible promoter (BBa_J33201) characterized by iGEM Edinburgh 2006. This part comprises of arsenic detoxification (ars) operon which is regulated by it’s repressor ArsR. Arsenic binds to the repressor ArsR and inhibits it’s ability to repress the promoter; subsequently resulting in the activation of promoter and expression of the gene present in the downstream region of the promoter. (8) We used Goniopora tenuidens GFP-like chromoprotein as our reporter gene. It is present in Goniopora tenuidens and is lilac in color.
We choose to work with PcadA (BBa_K1724000) which was characterized by iGEM SCUT 2015. It is a cadmium inducible promoter that is tightly regulated by a mutated version of MerR repressor protein. When the concentration of cadmium ions is high in the cell, cadmium ions bind with MerR and activate the CadA promoter by blocking its inhibition by MerR. We used blue chromoprotein amilCP (BBa_K592009); a part entered by iGEM Uppsala Sweden 2011.
We extracted nickel inducible promoter PnrsB from nrsBACD operon; a transporter expressing operon, of Synechocystic sp. PCC 6803. (10) We are using GFP like fluorescent chromoprotein amFP486 which is present in Anemonia manjano and functions as a green pigment protein.(11)
Our mercury device consists of a mercury inducible promoter PmerT (BBa_K346002) that was characterized by iGEM Peking 2010. It is part of the mercury resistant operon mer present in Tn21. It is regulated by a regulatory protein mer (12). We placed mer under a constitutive promoter. We are using Anemonia sulcata GFP-like chromoprotein FP595; a pigment protein that is present in Anemonia sulcata and functions as an intense purple pigment protein, as our reporter gene. (13)
We used CueR regulated copper inducible promoter (BBa_K190017) which was characterized by iGEM Groningen 2009. It’s transcription regulatory protein CueR activates transcription when cellular level of copper rises. (14) We are using GFP-like fluorescent chromoprotein FP538 that is present in Zoanthus sp. (Green polyp) and gives yellow color, as our reporter gene.(11)
Our zinc detection device comprises of a zinc inducible promoter smtA; part of the smtA gene in Synechococcus PCC7942. (15) We used spisPink chromoprotein (BBa_K1033925) which is present in a coral Stylophora pistillata and has been characterized by iGEM Uppsala 2013. (16)
The bio-available nature of heavy-metals provides them an ability to accumulate in fishes, mainly in the gill and the liver tissues. (17) This property of fish can be exploited to transform them into an ideal bio-reporter for the detection of heavy-metal contamination in aquatic environment through the principles of Synthetic Biology. (18)(19) The expression of a colorimetric reporter system can be used to assess the water-quality. Carvan et al. have elucidate the use of Zebra-fish, an aquatic model organism, for the detection of heavy-metals in in water. (20)
We are developing eukaryotic bio-bricks for the detection of heavy-metals in water. These bio-bricks will, prospectively, be used to develop a genetically engineered fish, which will express chromo-proteins in its specific regions. Due to safety guidelines of iGEM, these bio-bricks will not be directly introduced in fishes. We have first tested these devices in E.coli to assess their expression ability and then will move to a yeast model to analyze and compare the working of the bio-bricks in both Eukaryotic and Prokaryotic organisms. This will help in optimizing our assembly to develop the most efficient and effective bio-reporter system for the detection of heavy-metals in water.
The bio-reporter organism will be deployed at fish-farms where fishes continuously die as a result of the bio-accumulation of heavy-metals inside them. This can cause staggering financial losses to farm owners or those who deal in fishery.
7.Nielsen, Jens, and Jay D. Keasling. "Synergies between synthetic biology and metabolic engineering." Nature biotechnology 29.8 (2011): 693-695.8.Chen, J. and B.P. Rosen, Biosensors for inorganic and organic arsenicals. Biosensors, 2014. 4(4): p. 494-512.9.Gurskaya, N.G., et al., GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS letters, 2001. 507(1): p. 16-20.10.Englund, E., F. Liang, and P. Lindberg, Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Scientific reports, 2016. 6: p. 36640.11.Matz, M.V., et al., Fluorescent proteins from nonbioluminescent Anthozoa species. Nature biotechnology, 1999. 17(10).12.Brown, N.L., et al., The MerR family of transcriptional regulators. FEMS microbiology reviews, 2003. 27(2-3): p. 145-163.13.Lukyanov, K.A., et al., Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. Journal of Biological Chemistry, 2000. 275(34): p. 25879-25882.14.Outten, F.W., et al., Transcriptional Activation of an Escherichia coliCopper Efflux Regulon by the Chromosomal MerR Homologue, CueR. Journal of Biological Chemistry, 2000. 275(40): p. 31024-31029.15.Nivens, D.E., et al., Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring. Journal of Applied Microbiology, 2004. 96(1): p. 33-46.16.Alieva, N.O., et al., Diversity and evolution of coral fluorescent proteins. PLoS one, 2008. 3(7): p. e2680.17.Vinodhini, R., and M. Narayanan. "Bioaccumulation of heavy metals in organs of fresh water fish Cyprinus carpio (Common carp)." International Journal of Environmental Science & Technology 5.2 (2008): 179-182.18.Pawar, Nilambari, et al. "Development of a fluorescent transgenic zebrafish biosensor for sensing aquatic heavy metal pollution." Transgenic research 25.5 (2016): 617-627.19.Weiss, Ron. "Mammalian synthetic biology: from parts to modules to therapeutic systems." TRANSGENIC RESEARCH. Vol. 25. No. 2. VAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDS: SPRINGER, 2016.20.Carvan, Michael J., Tisha King Heiden, and Henry Tomasiewicz. "The utility of zebrafish as a model for toxicological research." Biochemistry and molecular biology of fishes 6 (2005): 3-41.