Difference between revisions of "Team:HokkaidoU Japan/Aggregation"

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<div id="Overview"><img src="https://static.igem.org/mediawiki/2016/f/fa/T--HokkaidoU_Japan--overview.png"  
 
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<br> 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 <i>Escherichia coli</i> (<i>E.coli</i>) will acquire the ability to produce the large amount of the SAP constantly.
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<br>  First of all, the way to continue producing the SAP in <i>E.coli</i> 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 <i>E.coli</i> to emit the SAP out of cytoplasm without accumulation (Fig1).
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<br>  Therefore, we expect that the emission of the SAP with TolC system contributes to inventing the new method for production of the hydrogel.
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<br>  When you harvest <span style="font-style: italic">E. coli</span> on your laboratory, is there a efficiently way to aggregate <span style="font-style: italic">E. coli</span> with the SAP?  We would like to try to suggest a possibility of the expression of <span style="font-style: italic">E. coli</span> aggregation by expression of the SAP fused to <span style="font-style: italic">E. coli</span> flagellum. Perhaps, a new method for the <span style="font-style: italic">E. coli</span> aggregation, except for Ag43 which has been adopted by some iGEM teams[1][2], may be found.
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<p>Fig1. Image of transportation with TolC system.</p>
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<p>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.</p>
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<td style="border-style:none; float:center"><img src="https://static.igem.org/mediawiki/2016/0/0a/T--HokkaidoU_Japan--futurework_image3.png"  
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width="500px" height="auto" alt="aggregation"></td>  
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<td style="border-style: none"; align="center"><span class="small">Fig. 1. Image of aggregation using SAP
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<br><span style="font-style: italic">E. coli</span> are aggregated by the binding through SAP. SAP is expressed on the fllagerum.</span></td>
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<br> 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.
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<br>FliC is an important domain which forms the filament (Fig. 2)[3]. It is known that any protein can be inserted into its domain without losing the function of the flagella. So we think that the aggregation of <span style="font-style: italic">E. coli</span> because of the bindings between the flagella is likely to happen by inserting SAP, for example, RADA16-I and P<span class="sitatuki">11</span>-4. Since it has been reported that the overexpression of the Ag43 decreases the survival rate of <span style="font-style: italic">E. coli</span>, we have to search for an alternative method for the aggregation of <span style="font-style: italic">E. coli</span>. 
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<br>Therefore we propose that inserting the SAP has an advantage of improving the efficiency of the consecutive chemical reactions when <span style="font-style: italic">E. coli</span> which have different functions approach each other.
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<p>Fig3. Construct for transporting SAP</p>
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<p>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.</p>
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<td style="border-style: none"; align="center"><span class="small">Fig. 2. Image of filament of <span style="font-style: italic">E. coli</span> 
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<br>Any protein can be inserted into the FliC.</span></td>
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<br>  If the method for mass production of the SAP inexpensively and on an industrial scale with <i>E.coli</i> 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.  
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<br>  If the method for industrial production of self-assembling peptides by using <span style="font-style: italic">E. coli </span> is established, it will contribute to disperse the utilization of hydrogel. To achieve this, we should focus on the synthesis and transportation of self-assembling peptides. We mentioned about “Transportation” above. Furthermore, we hope that other teams make further developments regarding “Synthesis”.And also the aggregation methods for <span style="font-style: italic">E. coli </span> in lab may be improved in the aspects of the cost or efficiency. From this aspects, the simpler methods without utilizing Ag43, His- tag, or RADA16-I are desirable. We expect that the understanding about the differences of the aggregating efficiency among the methods utilizing these proteins will be expanded. In addition, we hope that the aggregation method for <span style="font-style: italic">E. coli </span> by utilizing the self-assembling peptides we proposed will be useful in the future.
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<br>[1] http://parts.igem.org/Part:BBa_K554009
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[2] https://2015.igem.org/Team:WLC-Milwaukee/Project
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[1] <a href="https://2012.igem.org/Team:HokkaidoU_Japan/Project/Aggregation">HokkaidoU Japan 2012</a>
 
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[3]http://parts.igem.org/Part:BBa_K1166002https://2011.igem.org/Team:Harvard/Results/Biobricks#TolC_onehybrid_system _selection_strain_.28BBa_K615003.29
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[2] <a href="https://2014.igem.org/Team:Aberdeen_Scotland/Parts">Aberdeen Scotland 2014</a>
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<br>[3] Kuwajima, G. 1988. Construction of a minimum-size functional flagellin of Escherichia coli. J. Bacteriol. 170:3305-3309.
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Latest revision as of 20:17, 19 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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overview

When you harvest E. coli on your laboratory, is there a efficiently way to aggregate E. coli with the SAP? We would like to try to suggest a possibility of the expression of E. coli aggregation by expression of the SAP fused to E. coli flagellum. Perhaps, a new method for the E. coli aggregation, except for Ag43 which has been adopted by some iGEM teams[1][2], may be found.
aggregation
Fig. 1. Image of aggregation using SAP
E. coli are aggregated by the binding through SAP. SAP is expressed on the fllagerum.





design

FliC is an important domain which forms the filament (Fig. 2)[3]. It is known that any protein can be inserted into its domain without losing the function of the flagella. So we think that the aggregation of E. coli because of the bindings between the flagella 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.
filament
Fig. 2. Image of filament of E. coli
Any protein can be inserted into the FliC.





conclusion

If the method for industrial production of self-assembling peptides by using E. coli is established, it will contribute to disperse the utilization of hydrogel. To achieve this, we should focus on the synthesis and transportation of self-assembling peptides. We mentioned about “Transportation” above. Furthermore, we hope that other teams make further developments regarding “Synthesis”.And also the aggregation methods for E. coli in lab may be improved in the aspects of the cost or efficiency. From this aspects, the simpler methods without utilizing Ag43, His- tag, or RADA16-I are desirable. We expect that the understanding about the differences of the aggregating efficiency among the methods utilizing these proteins will be expanded. In addition, we hope that the aggregation method for E. coli by utilizing the self-assembling peptides we proposed will be useful in the future.

reference

[1] HokkaidoU Japan 2012
[2] Aberdeen Scotland 2014
[3] Kuwajima, G. 1988. Construction of a minimum-size functional flagellin of Escherichia coli. J. Bacteriol. 170:3305-3309.