Team:Ionis Paris/Parts

Our biosensor

Part Name Nickname Type Designer Lengh
BBa_K2023001 Biosensor device BB123 Measurement C.Chenebault, C.Soucies, B.Piot 3261 bp

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.

Favorite Parts

Part Name Nickname Type Designer Lengh
BBa_K2023004 Pr-RBS BB1 Composite C.Chenebault 430 bp
BBa_K2023005 XylR BB2 Coding C.Soucies 1704 bp
BBa_K2023006 Term-Pu-RBS-Gluc-term BB3 Composite B.Piot 1113 bp

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.

Part Name Nickname Type Designer Lengh
BBa_K2023002 XylR coding device BBA Regulatory C.Chenebault 2243 bp
BBa_K2023003 Gluc coding device with Pu BBB Reporter C.Soucies 1010 bp

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.

Parts for CelloCad

Part Name Nickname Type Designer Lengh
BBa_K2023014 GFP coding device with Pr C2 Device C.Chenebault 1293
BBa_K2023015 XylR coding device-GFP coding device with Pu C3 Measurement C.Soucies 3454 bp
BBa_K2023017 Pu-RBS C4 Composite C.Chenebault 340 bp
BBa_K2023016 GFP coding device with Pu C45 Reporter C.Soucies 1203 bp

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.

Gaussia derived parts

Part Name Nickname Type Designer Lengh
BBa_K2023007 Gluc coding device with Pr G1 device B.Piot 1100 bp
BBa_K2023008 Gluc-term G2 Composite C.Soucies 664 bp
BBa_K2023009 Gluc codon optimized G3 Coding B.Piot 561 bp

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.

XylR derived parts

Part Name Nickname Type Designer Lengh
BBa_K2023010 XylR with His tag X2 Coding C.Soucies 1728
BBa_K2023011 XylR-His tag coding device X3 Regulatory C.Soucies 2301 bp
BBa_K2023012 BB1- XylR-His tag BB12His Composite C.Chenebault 2164 bp
BBa_K2023013 XylR coding device with mRFP X1 Regulatory B.Piot 2975 bp

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.

Primers used to create our BioBricks

Primers used to sequence our BioBricks

Sequencing results are available on the registry.