Team:HokkaidoU Japan/Future work
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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, which has been used many times by the past iGEM teams, 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.
On the other hand, when you harvest E.coli on your laboratory, is there the way to aggregate E.coli efficiently with the SAP? We try to suggest the expression of the SAP on E. coli flagellum. Perhaps, a new method for the E.coli aggregation, except for Ag43 which has been adopted by some iGEM teams, may be found.
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.
On the other hand, FliC is an important domain which forms the filament (Fig4). It is known that any protein can be inserted into its domain without losing the function of the flagellar. So we think that the aggregation of E.coli because of the bindings between the flagellar is likely to happen by inserting SAP, for example, RADA16-I and P11-4. Since it has been reported that the overexpression of the Ag43 decreases the survival rate of E.coli, we have to search for an alternative method for the aggregation of E.coli.
Therefore we propose that inserting the SAP has an advantage of improving the efficiency of the consecutive chemical reactions when E.coli which have different functions approach each other.
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.
Besides, there may still be room for improvement for the financial aspect or the efficiency regarding the way to harvest E.coli in the laboratory. And a more simple method would be much more desirable without any expensive biological device. At last we wish that the research will be more conducted of the difference between the efficiency of the aggregation of E.coli with the biological tools, for example Ag43, RADA16-I, and P11-4. And we also desire for the possibility of the aggregation of E.coli with the SAP we suggest.
ここに本文
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, which has been used many times by the past iGEM teams, 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.
On the other hand, when you harvest E.coli on your laboratory, is there the way to aggregate E.coli efficiently with the SAP? We try to suggest the expression of the SAP on E. coli flagellum. Perhaps, a new method for the E.coli aggregation, except for Ag43 which has been adopted by some iGEM teams, may be found.
Fig2. Image of aggregation using SAP
E.coli are aggregated by the binding through SAP. SAP is expressed on the fllagerum.
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.
On the other hand, FliC is an important domain which forms the filament (Fig4). It is known that any protein can be inserted into its domain without losing the function of the flagellar. So we think that the aggregation of E.coli because of the bindings between the flagellar is likely to happen by inserting SAP, for example, RADA16-I and P11-4. Since it has been reported that the overexpression of the Ag43 decreases the survival rate of E.coli, we have to search for an alternative method for the aggregation of E.coli.
Therefore we propose that inserting the SAP has an advantage of improving the efficiency of the consecutive chemical reactions when E.coli which have different functions approach each other.
Fig4. Image of E.coli's firament
Any protein can be inserted into the FliC.
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.
Besides, there may still be room for improvement for the financial aspect or the efficiency regarding the way to harvest E.coli in the laboratory. And a more simple method would be much more desirable without any expensive biological device. At last we wish that the research will be more conducted of the difference between the efficiency of the aggregation of E.coli with the biological tools, for example Ag43, RADA16-I, and P11-4. And we also desire for the possibility of the aggregation of E.coli with the SAP we suggest.
ここに本文
