Difference between revisions of "Team:Goettingen/Results"

 
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<a href="" onClick=" $('#menu6').slideToggle(300, function callback() {  }); return false;"><h1>Scaffoldin Purification</h1></a>
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    <strong>Scaffoldin purification</strong>
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    After induction according to the methods collection, the targeted ScaA scaffoldin was purified by nickel affinity chromatography using Ni-NTA-agarose columns in a Äkta prime system (Fig. 1A). Following the addition of Imidazol two peaks were obtained. The fractions of interest were visualized in a SDS-PAGE gel (Fig. 1B). The gel showed that only the second fraction presents the expected size of 111kDa. However some other bands appeared along this fraction, therefore in order to obtain even more pure protein a Size Exclusion Chromatography (SEC) was done.
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[[File:Histag_purification_iGEM_Goettingen2015.jpeg|600px|thumb|center|]]
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    The SEC was done with a HiLoad Superdex 200 16/600 column (GE Healthcare) (Fig. 2A). SDS gel shows that a high concentration of the desired protein was
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    obtained and most of the previous contaminating bands were eliminated. Nevertheless there was an unknown band of low weight present in every fraction,
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    apparently it could be a second protein from E. coli with some affinity to the Scaffoldin than is then is co-purified with it (Fig. 2B).
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[[File:SEC_iGEM_Gottingen2015.jpeg|600px|thumb|center|]]
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  Future plans include to incubate the Scafoldin with the purified enzyme-dockerin construct in order to prove that the Flexosome can assembly by itself.
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<a href="" onClick=" $('#menu7').slideToggle(300, function callback() {  }); return false;"><h1>Copurification</h1></a>
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    Copurification of Scaffoldin and Enzyme-Dockerin
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    The main peaks after gelfiltration from the scaffoldin CTHE (see results scaffoldin) and CellulaseCCEL (Fig.1) were pooled and concentrated.
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[[File:TeamGoettingen_GeFi_CellulaseCCEL.jpg|650px|centre|thumb|B1-B3 were pooled for copurification.]]
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    They were then mixed and incubated together at 37°C for 15 min with agitation.
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    The solution was then used in another gelfiltration (Fig.2). The resulting peaks were run on a SDS-gel (Fig.3) and checked for cellulase activity.
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[[File:TeamGoettingen_Copurification_CTHE_CellulaseCCEL.jpg|650px|centre|thumb|Fig.2) The peak at A5-A7 may be the bound scaffoldin and dockerin, whilst the unbound  proteins are expected between B3 and C9.]]
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    The first peak from the copurification showed a distinct cellulase activity. As the peak ran considerably higher than expected than either the scaffoldin
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    or the CellulaseCCEL in the gelfitration, it can be expected that this peak contains a successfully formed complex between CellulaseCCEL and the CTHE
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    scaffoldin.
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<a href="" onClick=" $('#menu8').slideToggle(300, function callback() {  }); return false;"><h1>Giant Jamboree: Poster and Presentation</h1></a>
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Did you missed our presentation? Do you want to see our poster again?
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No problem, here you can find the files:
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[[Media:IGEM-presentation_final_team_Goettingen.ppt]]
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[[Media:Poster_teamGoettingen_2015.pdf]]
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Latest revision as of 22:03, 20 October 2015



Project Results

Transformation Efficiency Kit, RFP construct (iGEM)

RFP

Esterase and Phosphatase

Cellulase

BioBricks

Scaffoldin Purification

Copurification

Giant Jamboree: Poster and Presentation