Those 3 parts are probably the most important because they are the fondations of the biosensor right above. We designed and ordered the sequences to Integrated DNA Technologies. We first assembled BB1 and BB2 in pSB1C3 to create BB12 and then we had BB3 in order to produce BB123 the biosensor. The Pr promoter (from BB1) is found in the toluene recognition system and is composed of 410 bp. This promoter without the RBS is available on the iGEM registry at this ID access: BBa_I723018. In fact, we chose to use this promoter because it is the specific promoter for the XylR gene. This promoter is naturally constitutive. It leads to the permanent production of the XylR protein in the cells. Indeed, the induced luminescence has to be proportional to the pollutant rate. Therefore, when pollutant molecules enter in the bacteria, the XylR protein should be present in sufficient amount to capture all of it. The XylR gene, encodes for the XylR protein and is regulated by the Pr promoter in its native context. This XylR gene is available on the iGEM Registry of Standard Biological Parts (BBa_K1834844). The XylR that we designed for our biosensor (BB2) is a bit different because it is optimized for E.Coli DH5-Alpha. The XylR protein, mined from Pseudomonas putida, is involved in the transcriptional activation of the toluene recognition system. This regulatory protein allows the detection of aromatic hydrocarbons that carry a methyl group, i.e. xylene, toluene and 1-chloro-3-methyl-benzene. The A domain of the XylR protein (sensing domain), binds to the pollutant molecule. This leads to the formation of a tetramer. The C domain is involved in the dimerization of XylR, which is ATP dependent. The made up tetramer acts as an activator transcriptional factor for the Pu promoter through the DNA binding D domain. Pu is a promoter found in the toluene recognition system and is composed of 320 bp. This promoter is available on the iGEM registry at this ID access: BBa_I723020. We chose to use this promoter because of its sensibility to the transcriptional regulator XylR bound to xylene, toluene or 1-chloro-3-methyl-benzene. GLuc gene is found in a well-known organism, the copepod Gaussia princeps. It encodes for the Gaussia luciferase enzyme, also known as GLuc, which is involved in a bioluminescence process. This enzyme degrades coelenterazine, into a product, celenteramide. With an optimal substrate level, this step produces energy in the form of light that can be detected with a fixed spectrophotometer at 488nm. We chose to use the GLuc-His part, a gene of 522 bp, available on the iGEM registry at the (BBa_K1732027) which codes for the Gaussia luciferase followed by 6 histidines and optimized it for E.coli: ID adress of our GLuc-His: Bba_K2023009. In our plasmid, this gene is positioned after the Pu promoter, to report the activation of the toluene recognition system. The Gaussia luciferase needs the addition of substrate to ensure its activity because this molecule is not synthetized by our biosensor. Therefore, in the laboratory, luciferase substrate can be added at the same time in each sample ensuring that every measurement will be taken at the same time. This allow a better consistency between our different results. Also, due to its secreted form, lysing cells in order to assay GLuc activity is not necessary. The Gaussia luciferase is an ideal reporter gene because of its stability at high temperature thanks to disulfide bonds and because it has extremely high activity in light production for very sensitive assays. When compared to Firefly and Renilla luciferase, GLuc generates over 1000-fold higher bioluminescent signal intensity[1][2]. The NanoLuc has an activity a little higher but this luciferase is very recent and thus less characterized. Advantages of luminescence, over fluorescence, include the absence of background noise, the amplification of signal and a high dynamic range that spans many orders of magnitude. Indeed, since light emission depends strictly on the chemical reaction between the substrate and the luciferase, there is no background noise originating from the sample [3]. Furthermore, the turnover of the light reaction significantly amplifies the reporter signal. Even though bioluminescence is currently used mainly for transcription study and cell imaging, this method become increasingly popular for quantitative analysis. Even though bioluminescence is currently used mainly for transcription study and cell imaging, this method become increasingly popular for quantitative analysis. [3] For more information about bioluminescence and the Gaussia luciferase, click here ! [1] Inouye, S., Sahara-Miura, Y., Sato, J., Iimori, R., Yoshida, S., and Hosoya, T. (2013). Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: Comparison of substrate specificity for C2-modified coelenterazines. Protein Expression and Purification 88, 150–156. [2] S. Inouye, Y. Sahara, Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps, Biochem. Biophys. Res. Commun. 365 (2008) 96–101 [3]Wood, K (2011), The bioluminescence advantage, laboratorynews, available on: http://www.labnews.co.uk/features/the-bioluminescence-advantage-13-09-2011/