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Team:Penn/SurfaceDisplayBBa
From 2012.igem.org
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| - | We sought to create a system in which iGEM teams and labs can display any protein of their choosing on the surface of E. coli. We engineered a novel Suface Display BioBrick (BBa K811004) using the N and C terminal domains of the Ice Nucleation Protein (INPNC) which allows iGEM teams to fuse any desired protein to INPNC using a simple ligation protocol with BamHI and PstI restriction sites.</p> | + | We sought to create a system in which iGEM teams and labs can display any protein of their choosing on the surface of E. coli. We engineered a novel Suface Display BioBrick (BBa K811004) using the N and C terminal domains of the Ice Nucleation Protein (INPNC), which allows iGEM teams to fuse any desired protein to INPNC using a simple ligation protocol with BamHI and PstI restriction sites.</p> |
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| - | As a preliminary proof | + | As a preliminary proof of concept for our INPNC-enabled system, we displayed the red fluorescent protein mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).</p> |
<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/a2/MCherry-vs-INPNC-mCherry.jpg" width="250" height="350"/><br> | <div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/a/a2/MCherry-vs-INPNC-mCherry.jpg" width="250" height="350"/><br> | ||
| - | <b>Figure 1</b></div>Figure 1: Surface | + | <b>Figure 1</b></div>Figure 1: Surface display of mCherry using INPNC system. INPNC-mCherry and Intein-mCherry fusions were expressed in E. coli BL21 in the pET26b expression vector and Wood-Intein expression plasmid, respectively. When fused to INPNC, almost all mCherry was localized in the membrane fraction after sonication and centrifugation, while in the case of Intein-mCherry, all mCherry was localized in the cytoplasmic lysate.</div> |
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Revision as of 00:00, 27 October 2012
Generalized Surface Display System
We sought to create a system in which iGEM teams and labs can display any protein of their choosing on the surface of E. coli. We engineered a novel Suface Display BioBrick (BBa K811004) using the N and C terminal domains of the Ice Nucleation Protein (INPNC), which allows iGEM teams to fuse any desired protein to INPNC using a simple ligation protocol with BamHI and PstI restriction sites.
As a preliminary proof of concept for our INPNC-enabled system, we displayed the red fluorescent protein mCherry on the outer membrane of E. coli BL21. After sonication and centrifugation of induced cells, almost all of the mCherry was localized in the membrane fraction when fused to INPNC, whereas in the control Intein-mCherry fusion (which exhibits cytoplasmic localization), all of the mCherry was contained in the lysate (Figure 1).
Figure 1
