Team:INSA-UPS France/Parts

Parts



*** All parts listed here are available upon request ***

Feel free to contact us, if you want to know more about our parts: igem.toulouse@gmail.com

New parts submitted to the registry

Name Function Type Part State
BBa_K2278001 QS molecule generator basic working
BBa_K2278002 QS molecule generator basic not tested
BBa_K2278011 Diacetyl generator basic issues
BBa_K2278021 D-NY15 AMP generator basic working
BBa_K2278022 Leucrocin I AMP generator basic unsuccessful
BBa_K2278023 coT2 AMP generator basic unsuccessful




Existing Parts we have contributed to characterized



BBa_J04450: RFP coding device

Figure 1: BBa_J04450 biobrick conjugated in Vibrio harveyi.

BBa_J04450 was tested in the Vibrio harveyi background. The biobrick was cloned in a broad host range plasmid (pBBR1MCS-4) and conjugated into Vibrio harveyi to demonstrate the production of RFP in this chassis.

To learn more: see BBa_J04450 and our results.



BBa_K431009: glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP)

Figure 2: construction to characterized BBa_K431009.

In this part encoding sequence of D-NY15 was placed under the yeast constitutive promoter pGAP. This part was characterized by RT-qPCR.

To learn more: see BBa_K431009 and our results.





BBa_K1800001: alpha factor secretion signal

Figure 3: construction to characterized BBa_K1800001.

The α-factor sequence contains a kozak region and a signal sequence to secrete the produced peptides. The functionality of the signal factor was investigated by demonstrating that AMP are present in the supernatant using a toxicity assay.

To learn more: see BBa_K180001 and our results.



Pichia pastoris complete module with reporter gene : Odr-10 diacetyl receptor (BBa_K1072010), pFUS1 (BBa_K1072023)

This part includes Odr-10 receptor under the control of pGAP, a constitutive yeast promoter. When diacetyl binds to Odr-10 a cascade of activation of Ste proteins (endogenous to P. pastoris ) will lead to the binding of Ste12 on pFUS1 promoter, and the expression of RFP should be activated.

This system allowed to produce RFP in response to diacetyl.

This construction was cloned in pPICZalpha yeast vector.

To learn more: see BBa_K1072010, BBa_K1072023 and our results.





Parts used in our project but not submitted to the registry



Vh1: First part of Vibrio harveyi gene circuit

Figure 4: first part of Vibrio harveyi gene circuit (Vh1).

This part contains the first half of Vibrio harveyi quorum CqsS receptor which is able to detect CAI-1 family molecule. The receptor is under the control of a constitutive promoter.

This part has been designed due to IDT gBlocks synthesis requirements. The ordered gBlocks size should not exceed 3kb (the total length of the Vibrio harveyi circuit is 6929 bp). The XhoI restriction site naturally present in the gene was used reassemble the cqsS* sequence.

Therefore, it was impossible to use pSB1C3 vector because of the presence of a XhoI restriction on the vector. This part was then subcloned in pBR322.

To learn more: see our design, and our results.



Vh 2 : Second part of Vibrio harveyi gene circuit

Figure 5: second part of Vibrio harveyi gene circuit (Vh2).

This part contains the second half of Vibrio harveyi quorum CqsS* receptor which is able to detect CAI-1 family molecule. It also contains the tetR repressor under the control of pQRR4 promoter.

This part has been designed due to IDT gBlocks synthesis requirements. The ordered gBlocks size should not exceed 3kb (the total length of the Vibrio harveyi circuit is 6929 bp). The ApaI (not present on pSB1C3 vector) and XhoI restriction (naturally present in the cqsS* gene) were used to reassemble the synthetic circuit.

To learn more: see our design, and our results.



Vh 1+2: Vibrio harveyi invertor system

Figure 6: Vibrio harveyi invertor system (Vh 1+2).

This part includes the complete cqsS* gene under a constitutive promoter and the tetracyclin repressor under the control of pQRR4 promoter which activation depends on the detection of quorum sensing inducers by CqsS*.

Vh1+2 is the assembly of Vh2 in Vh1. This part was cloned in pBR322.

To learn more: see our design, and our results.



Vibrio harveyi complete gene circuit

Figure 7: Vibrio harveyi complete gene circuit.

This part includes the complete cqsS under the control of a constitutive promoter and the tetracycline repressor (TetR) under the control of pQRR4 promoter which activation depends on CqsS* recognition of quorum sensing inducers. The als gene, implied in the diacetyl production pathway, is under the control of pTet which can be repressed by TetR.

This part is the assemblage of Vh1+2 in BBa_K2278011. It allows to produce diacetyl is produced upon detection of Vibrios CAI-1 quorum sensing molecule. The als gene is flanked with two restriction enzyme sites to clone a reporter gene such as rfp.

The assembly of this part was not released due to technical and time issues.

To learn more: see our design, and our results.



Pichia pastoris D-NY15 AMP generator

Figure 8: Pichia pastoris D-NY15 AMP generator.

This system allowed us to constitutively express BBa_K2278021 with a yeast promoter. This system was used to produce AMP at the high concentration.

The α-factor sequence contains a RBS and a signal sequence to secrete the produced peptides.

The restriction enzyme sites allow to extract individually each components of the plasmid. This construction was cloned in pPICZalpha yeast vector.

To learn more: see BBa_K2278021, and our results.



Pichia pastoris Leucrocin I AMP generator

Figure 9: Pichia pastoris Leucrocin I AMP generator.

This system allowed us to constitutively express the BBa_K2278022 with a yeast promoter. This system was used to produce AMP at high concentration.

The α-factor sequence contains a RBS and a signal sequence to secrete the produced petides.

The restriction enzyme sites allow to extract individually each components of the plasmid. This construction was cloned in pPICZalpha yeast vector.

To learn more: see BBa_K2278022 and see our results.



Pichia pastoris cOT2 AMP generator

Figure 10: Pichia pastoris cOT2 I AMP generator.

This system allowed us to constitutively express BBa_K2278023 with a yeast promoter. This system was used to produce AMP at high concentration.

The α-factor sequence contains a RBS and a signal sequence to secrete the produced peptides.

The restriction enzyme sites allow to extract individually each components of the plasmid. This construction was cloned in pPICZalpha yeast vector.

To learn more: see BBa_K2278023 and our results.



Pichia pastoris complete module

Figure 11: Pichia pastoris complete module

This system allowed us to constitutively express Odr10 receptor to detect diacetyl at any time. Odr10 pathway leads to the activation of pFUS promoter when diacetyl is present in the environment, hence the AMP are produced.

The α-factor signal and coT2 gene are flanked with two restriction enzyme sites to clone a reporter gene such as RFP. This construction was cloned in pPICZalpha yeast vector.

To learn more: see BBa_K2278023, and our results.