Team:HokkaidoU Japan/Aggregation
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Team:HokkaidoU Japan
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In this Jamboree, we focused on the self-assembling peptide (SAP). This peptide has been used as the hydrogel in the medical area. We hope that Escherichia coli (E.coli) will acquire the ability to produce the large amount of the SAP constantly.
First of all, the way to continue producing the SAP in E.coli needs to be established. As our Future work, we want to propose that TolC-Transportation-System[1], which has been used many times by the past iGEM teams[2][3], enables E.coli to emit the SAP out of cytoplasm without accumulation (Fig1).
Therefore, we expect that the emission of the SAP with TolC system contributes to inventing the new method for production of the hydrogel.
Induced promoter triggers the upstream expression of HlyB, HlyD and TolC. When the target protein is passed through outer membrane, these three proteins play the role as one unit on the membrane. Furthermore, the HlyA adds the signal sequence to the complex (HisTag-RADA16-I) and guides it to TolC. After the complex passes through TolC, it can be harvested by the affinity purification.
If the method for mass production of the SAP inexpensively and on an industrial scale with E.coli is established, it will contribute for the spread of utilization of the hydrogel. We should consider producing and transporting the hydrogel separately. Although we mention the latter as our Future work in this Jamboree, we also hope any other team to arise the new findings or the development about the former as well.
[1] http://parts.igem.org/Part:BBa_K554009
[2] https://2015.igem.org/Team:WLC-Milwaukee/Project
[3]http://parts.igem.org/Part:BBa_K1166002https://2011.igem.org/Team:Harvard/Results/Biobricks#TolC_onehybrid_system _selection_strain_.28BBa_K615003.29
In this Jamboree, we focused on the self-assembling peptide (SAP). This peptide has been used as the hydrogel in the medical area. We hope that Escherichia coli (E.coli) will acquire the ability to produce the large amount of the SAP constantly.
First of all, the way to continue producing the SAP in E.coli needs to be established. As our Future work, we want to propose that TolC-Transportation-System[1], which has been used many times by the past iGEM teams[2][3], enables E.coli to emit the SAP out of cytoplasm without accumulation (Fig1).
Therefore, we expect that the emission of the SAP with TolC system contributes to inventing the new method for production of the hydrogel.
Fig1. Image of transportation with TolC system.
SAP is produced, binding HlyA and HisTag in the cytoplasm. The short amino chain is a kind of the marker and SAP is guided to TolC transporter. After then, SAP passes through it and is collected with Histag.
Induced promoter triggers the upstream expression of HlyB, HlyD and TolC. When the target protein is passed through outer membrane, these three proteins play the role as one unit on the membrane. Furthermore, the HlyA adds the signal sequence to the complex (HisTag-RADA16-I) and guides it to TolC. After the complex passes through TolC, it can be harvested by the affinity purification.
Fig3. Construct for transporting SAP
The induced promoter is needed if you want to produced and emit SAP, for exaple, PLac or PTet. We thought that the region which control TolC system and the one which control HisTag-SAP-HlyA should not be apart because the same promoter was more convenient to express.
If the method for mass production of the SAP inexpensively and on an industrial scale with E.coli is established, it will contribute for the spread of utilization of the hydrogel. We should consider producing and transporting the hydrogel separately. Although we mention the latter as our Future work in this Jamboree, we also hope any other team to arise the new findings or the development about the former as well.
[1] http://parts.igem.org/Part:BBa_K554009
[2] https://2015.igem.org/Team:WLC-Milwaukee/Project
[3]http://parts.igem.org/Part:BBa_K1166002https://2011.igem.org/Team:Harvard/Results/Biobricks#TolC_onehybrid_system _selection_strain_.28BBa_K615003.29
