Team:HokkaidoU Japan/Transport

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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overview

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 a 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 (like BBa_K554013), which has been used many times by the past iGEM teams[1][2], enables E. coli to emit the SAP out of cytoplasm without accumulation (Fig. 1).
Therefore, we expect that the emission of the SAP with TolC system contributes to inventing the new method for production of the hydrogel.

transport
Fig. 1. Image of transportation with TolC system.
SAP is produced, binding HlyA and His-tag 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 His-tag.





design

Induced promoter triggers the upstream expression of HlyB, HlyD and TolC. These three proteins form a complex buried in outer membrane and transport target proteins to bacterial surface across outer membrane. Furthermore, the HlyA adds the signal sequence to the complex (His-tag-SAP) and guides it to TolC. After the complex passes through TolC, it can be harvested by the affinity purification.

construct
Fig. 2. Construct for transporting SAP.
The induced promoter is needed if you want to produce and emit SAP, for exaple, PLac or PTet. We thought that the region which controls TolC system and the one which controls His-tag-SAP-HlyA should not be apart because the same promoter was more convenient to express.





conclusion

If the method for mass production of the SAP inexpensively and on an industrial scale with E. coli is established, it will contribute to 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.


reference

[1] UNICAMP-EMSE Brazil 2011
[2] Stanford 2009
[3] Utah State 2011

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