The biosensor is the heart of our project and it took us a lot of time to create. Like it is already explained in the Biobrick Design section, this biobrick is composed of the Pr promoter (BBa_K2023004) which is a constitutive promoter driving the transcription of the XylR gene (BBa_K2023005) coding for the XylR protein. The XylR protein is able to bind aromatic hydrocarbons that carry a methyl group like toluene and xylene. When XylR has bound to toluene for instance, it is able to regulate the Pu promoter has a transcriptional regulator. Then, Pu is activated and it allows the transcription of the bioluminescent reporter gene GLuc (Pu+GLuc: BBa_K2023003), coding for the Gaussia luciferase. When this enzyme reacts with its substrate luciferine, a substance called Coelenterazine, it emits luminescence.
Those 3 parts are probably the most important ones because they are the foundations of the biosensor shown 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. For more information on those parts click on the Biobricks to go to the registry.
BBA and BBB are the coding device that compose our biosensor. BBA is made with Pr-RBS-XylR-Term and can be linked with other reporter like the GFP reporter. BBB is composed of GLuc coding device with Pu: RBS-GLuc-Term-Pu.
Those parts allowed us to start the CelloCad side project. In order to compare the RPU of the Pr and the Pu promoter with the reference promoter BBa_J23101 we designed C2 and C3. To produce the part C3 we have needed the parts C4 and C45.
G1, G2 and G3 are 3 parts that come from the GLuc-His part of 522 bp (BBa_K1732027). G1 will allow a positive control during a future pollution quantification test. G2 is a useful part because it is the GLuc-His part with a terminator and it can be adapted with another detector than the XylR. G3 is the GLuc-His part that we optimized for the chassis E.Coli and for the IDT sequence order.
Those 4 parts are derived from the XylR gene that we optimized for our chassis E.coli. BBa_K2023010 (X2) is the XylR gene with a His Tag. BB12His is X2 plus the promoter Pr. The coding device is X3: it is composed of BB12His plus a terminator. Those BioBricks will help future iGEM team working with XylR to better characterize this part as they would be able to quantify and purify it.
In the same way of monitoring XylR synthesis, we designed XylR coding device with mRFP. This device is composed of Pr-RBS-XylR-RBS-mRFP-Terminator. It permits us to prove that the XylR is synthesized: colonies becomes red thanks to the mRFP.
Improvement of BBa_K1834844 (XylR)
We optimized the BioBrick BBa_K1834844 for a use in E.coli as well as for IDT synthesis. We added an His-Tag to this BioBricks to help characterization and purification. We called this optimized BioBrick BBa_K2023010 and designed it in order to purify and quantify XylR produce by bacteria prior to bioluminescence test.
