Team:INSA-UPS France/Parts

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Parts

New parts submitted to the registry

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




Existing Parts we have contributed to characterized



<partinfo>BBa_J04450</partinfo>: 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 <partinfo>BBa_J04450</partinfo> and our results.



<partinfo>BBa_K431009</partinfo>: glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP)

Figure 2: construction to characterized BBa_K431009.

The pGAP promoter was used as a constitutive promoter and was characterized by qRT-PCR using d-ny15 as reporter gene.

To learn more: see <partinfo>BBa_K431009</partinfo> and our results.





<partinfo>BBa_K1800001</partinfo>: 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 through a toxicity assay.

To learn more: see <partinfo>BBa_K180001</partinfo> and our results.



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

This part includes Odr-10 receptor driven by a constitutive yeast promoter. pFUS1 is triggered by the Ste12 protein activated by Odr-10 pathway when diacetyl is detected.

This system allowed to produce RFP in response to diacetyl.

This construction was cloned in pPICZalpha yeast vector.

To learn more: see <partinfo>BBa_K1072010</partinfo>, <partinfo>BBa_K1072023</partinfo> 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 driven by 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 driven by a constitutive promoter and the tetracyclin repressor under the control of pQRR4 promoter which activation depends on CqsS* detection of quorum sensing inducers.

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 gene driven by 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 highest concentration.

The pAOXI promoter ensures genome recombination in Pichia pastoris genome. 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 <partinfo>BBa_K2278021</partinfo>, 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 the highest concentration

The pAOXI promoter makes genome recombination easier in Pichia pastoris genome. 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 <partinfo>BBa_K2278022</partinfo> 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 the highest concentration

The pAOXI promoter makes genome recombination easier in Pichia pastoris genome. 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 <partinfo>BBa_K2278023</partinfo> 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 in case of diacetyl detection, 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 <partinfo>BBa_K2278023</partinfo>, and our results.