Team:NU Kazakhstan/E.Coli

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<center><h3>E.coli expression system</h3></center>
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<p style="line-height:200%;padding-top:10px">E.Coli is a gram negative bacteria which is widely used model organism for the expression of different products. We were planning to express streptavidin on the surface of E.coli using two expression systems. First is Lpp-OmpA system and second is INP expression system, both of which were obtained from 2013 distribution kit. The constructed parts were planned to be inserted in pET28a plasmid under the control of T7 promoter. Our constructs should look like the following:</p></div>
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<tr><td><img src="https://static.igem.org/mediawiki/2013/4/41/Lpp_omp.png" width="400" height="200"></td>
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<td><img src="https://static.igem.org/mediawiki/2013/f/f9/Inp.png" width="400" height="200"></td></tr>
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<tr><td><center><b>Lpp-OmpA expression sytem</b></center></td>
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<td><center><b>INP expression system</b></center></td</tr>
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<p style="line-height:200%;padding-top:10px">In this project, we were also planning to use CdSe/ZnS QDs for conjugation to aptamers to create biosensor of sandwich manner. Two different aptamers against carcinoembryonic antigen (CEA) would be used. The first aptamer would become attached to streptavidin via biotin, and the second aptamer, which would be conjugated to QDs, would bind to the target protein itself. After that, fluorescent spectroscopy could be used to detect binding between conjugated aptamers and the corresponding target protein.</p>
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<p style="line-height:200%;padding-top:10px">In order to see the specificity of binding between aptamers-QDs conjugates and the target, various imaging techniques such as fluorescence spectrophotometer, confocal laser microscopy, flow cytometry and other techniques can be applied.</p></div>
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Latest revision as of 16:00, 27 September 2013

NU_Kazakhstan

E.coli expression system

E.Coli is a gram negative bacteria which is widely used model organism for the expression of different products. We were planning to express streptavidin on the surface of E.coli using two expression systems. First is Lpp-OmpA system and second is INP expression system, both of which were obtained from 2013 distribution kit. The constructed parts were planned to be inserted in pET28a plasmid under the control of T7 promoter. Our constructs should look like the following:

Lpp-OmpA expression sytem
INP expression system

In this project, we were also planning to use CdSe/ZnS QDs for conjugation to aptamers to create biosensor of sandwich manner. Two different aptamers against carcinoembryonic antigen (CEA) would be used. The first aptamer would become attached to streptavidin via biotin, and the second aptamer, which would be conjugated to QDs, would bind to the target protein itself. After that, fluorescent spectroscopy could be used to detect binding between conjugated aptamers and the corresponding target protein.

In order to see the specificity of binding between aptamers-QDs conjugates and the target, various imaging techniques such as fluorescence spectrophotometer, confocal laser microscopy, flow cytometry and other techniques can be applied.

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