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Revision as of 03:10, 1 October 2016
Team:HokkaidoU Japan
![overview](https://static.igem.org/mediawiki/2016/f/fa/T--HokkaidoU_Japan--overview.png)
We advocate the new method to create multimer using the self-assembling ability of SAP. As fusing SAP to the end of protein, it will condense with other proteins’ SAP domains and form the complex. BY using this method, we’ll be able to connect several enzymes and allow huge complexed proteins formed. It’ll improve the efficiency of continuous reaction. The ordinary method uses linkers to connect proteins. We suggest this new method with SAP is superior to the ordinary one for these resons.
![enzymatic reaction](https://static.igem.org/mediawiki/2016/3/3b/T--HokkaidoU_Japan--multimerization_image6.png)
![large block](https://static.igem.org/mediawiki/2016/0/08/T--HokkaidoU_Japan--multimerization_image7.png)
Linker Method | SAP Method |
---|---|
It is difficult to connect several huge proteins | It is possible to synthesize the proteins individually. It also can form huge complex |
Regulated by one promoter | Each proteins can be expressed individually |
The possibility of deformation of 3D-structure | Low possibility of deformation since they connect with only proteins which can condense |
![linker methods](https://static.igem.org/mediawiki/2016/e/e0/T--HokkaidoU_Japan--multimerization_image1.png)
![SAP methods](https://static.igem.org/mediawiki/2016/7/72/T--HokkaidoU_Japan--multimerization_image2.png)
![steric hindrance](https://static.igem.org/mediawiki/2016/2/23/T--HokkaidoU_Japan--multimerization_image3.png)
![demerit](https://static.igem.org/mediawiki/2016/d/d8/T--HokkaidoU_Japan--multimerization_image5.png)
![resolution](https://static.igem.org/mediawiki/2016/c/cf/T--HokkaidoU_Japan--multimerization_image4.png)
There are also disadvantages using SAP. Since the number of the possible combination of several different is infinite, there is no guarantee that we can always obtain the expected combination when we form the protein complex. The one solution for the problem is limiting the number of combination by using different SAP. As forming protein complex with different functions, this multimer forming method with SAP let us create more functional unit. When same kind of proteins are used, it’ll be large block and its function is expected to be enhanced.
![methods](https://static.igem.org/mediawiki/2016/2/2c/T--HokkaidoU_Japan--methods.png)
We tried forming multimers using the self-assembling peptide, P11-4 and RADA-16. We Connected short linker(GGCGG) and SAP to both ends of the protein. In this experiment, we formed the multimers of GFP. GFP’s molecular mass is 26891Da. When fusing with P11-4, it’s 31709Da. With RADA, it’s 31943Da. When they form multimer, the molecular mass will be more than 60kDa. Consequently, we used the filter which filters out the proteins with more than 50KDa. For the evaluation, we ordered IDT the designed constructions and put them on the vectors. Then, we introduced them to E.Coli. Using IPTG induction , the proteins were expressed. Causing bacteriolysis with freeze-thaw, we acquired the supernatant contains the proteins by centrifugal separation. Purifying the protein with Ni-affinity chromatography, we filtrated the solution to separate the proteins with less than 50KDa. We irradiated 480nm light to filtrate and observed whether 580nm wave-length light was emitted.
![construct](https://static.igem.org/mediawiki/2016/d/df/T--HokkaidoU_Japan--multimerization_construct.png)
![methods](https://static.igem.org/mediawiki/2016/0/0c/T--HokkaidoU_Japan--multimerization_image8.png)
![results](https://static.igem.org/mediawiki/2016/d/de/T--HokkaidoU_Japan--results.png)
ここに本文
![conclusion](https://static.igem.org/mediawiki/2016/7/7b/T--HokkaidoU_Japan--conclusion.png)
ここに本文