Difference between revisions of "Team:HokkaidoU Japan/Multimerization"
| Line 6: | Line 6: | ||
<div id="Methods"><img src="https://static.igem.org/mediawiki/2016/2/2c/T--HokkaidoU_Japan--methods.png" | <div id="Methods"><img src="https://static.igem.org/mediawiki/2016/2/2c/T--HokkaidoU_Japan--methods.png" | ||
width="270px" height="90px" alt="methods"></div> | width="270px" height="90px" alt="methods"></div> | ||
| − | <p> | + | <p>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. | ||
| + | </p> | ||
<div id="Results"><img src="https://static.igem.org/mediawiki/2016/d/de/T--HokkaidoU_Japan--results.png" | <div id="Results"><img src="https://static.igem.org/mediawiki/2016/d/de/T--HokkaidoU_Japan--results.png" | ||
width="270px" height="90px" alt="results"></div> | width="270px" height="90px" alt="results"></div> | ||
Revision as of 01:56, 1 October 2016
"
Team:HokkaidoU Japan
ここに本文
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.
ここに本文
ここに本文
