=Characterization of the K1372001 biobrick from the 2014 "This is not a lemon" Paris Saclay project=
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As a part of the characterization of a previous existing Biobrick Part, we chose the BBa_K13372001 biobrick from the Paris Saclay 2014 project “This is not a lemon”. It was designed to mimic the ripening of a lemon in E.Coli by the salicylate inducible expression a t-RNA suppressor.
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The Paris Saclay 2014 team chose to use chromoproteins to express these colors in E. coli. Chromoproteins are reflective proteins that contain a pigmented prosthetic group and do not need to be excited to be seen. They fused a yellow chromoprotein with a blue one in order to display a green color. This construction is be referred as the green fusion chromoprotein. In order to make the bacteria ripe like a real lemon, then decided to take advantage of the designing of the fusion protein by using a translational suppression system. They added amber codons within the linker separating the yellow and the blue chromoproteins. Therefore, the expression of a suppressor t-RNA will suppress amber codons allowing the translation of the green fusion chromoprotein. Conversely, the down regulation of the suppressor t-RNA through time will allow bacteria switch from green to yellow, thus simulating the ripening of a real lemon. This system is referred to as the color switch system.
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[[File:T--Paris_Saclay--project_2014_1.jpeg.png|400px|thumb|centre|summary of the lemon ripening project]]
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The t-RNA used is the supD suppressor t-RNA. It has been placed under control of a salicylate inducible promoter Psal to suppress the introduced amber codon. The nahR gene encodes a transcriptional regulator that is induce by salicylate and thus bind nah or Psal promoters. In presence a of high salicylate concentration in the agar media supD will be expressed and so the green fusion chromoprotein: bacteria will display a green color. However, as bacteria grow into agar, less salicylate will remain available into the media. Thus, the decrease of the nahR-salicylate complex amount within bacteria will lead to supD downregulation through time. In turn, decrease of supD amount will result in less codon readthrough and so less translation of the green fusion protein and more translation of the yellow chromoprotein. As a result, bacteria will gradually change from green to yellow.
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[[File:T--Paris_Saclay--project_2014_2.jpeg|400px|thumb|centre|explanatory diagram of the lemon ripening]]
