Team:Penn/SurfaceDisplayBBa

From 2012.igem.org

(Difference between revisions)

Latest revision as of 03:28, 27 October 2012

Penn 2012 iGEM Wiki

Display Your Own Proteins: BBa_K811005

Surface display has wide applications in industry and medicine. We wanted to create a simple system for any iGEM team or lab to display proteins on the surface of bacteria. Using INPNC and our 12aa GS linker, we created INPNC-MCS, a BioBrick which allows teams to clone in any INPNC fusion partner of their choosing and have it displayed on the surface of bacteria. This system only requires a one step ligation with BamHI and PstI cloning sites. This BioBrick was recently awarded "Best BioBrick: Engineered" at the 2012 iGEM Americas East Regional Jamboree!

Experience

Part BBa_K811005 has been utilized by the 2012 Penn iGEM team to display a two completely different classes of proteins and we are currently using it to display a variety of other proteins. The red fluorescent protein mCherry has been successfully displayed on the surface of E. Coli, where it can produce fluorescence. We displayed 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

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.

Furthermore, a HER2 binding protein, DARPin H20-2-G3 has also displayed on the surface of E. Coli, and has been shown to retain its HER2 binding affinity upon surface display through Part BBa_K811005.

Retrieved from "http://2012.igem.org/Team:Penn/SurfaceDisplayBBa"

@@ Line 21: / Line 21: @@
 <div class="bigbox">
-<b><div class="name" align="center">Overview
+<b><div class="name" align="center">Display Your Own Proteins: BBa_K811005
 </div></b><br />
+<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/0/06/Your-Protein-Here.gif" width="500" height="500"/></div></div>
 <p style="color:black;text-indent:30px;">
-Ice nucleation protein (INP) is a protein found in <i>Xanthomonas campestris</i> pc. campestris BCRC 12846. Its function is to provide a surface for ice nucleation, which results in the formation of ice crystals. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurrence in prokaryotes.</p>
+Surface display has wide applications in industry and medicine. We wanted to create a simple system for any iGEM team or lab to display proteins on the surface of bacteria. Using INPNC and our 12aa GS linker, we created INPNC-MCS, a BioBrick which allows teams to clone in any INPNC fusion partner of their choosing and have it displayed on the surface of bacteria. This system only requires a one step ligation with BamHI and PstI cloning sites. <b>This <a href="http://partsregistry.org/Part:BBa_K811005">BioBrick</a> was recently awarded "Best BioBrick: Engineered" at the 2012 iGEM Americas East Regional Jamboree!</b> </p>
 </div>
@@ Line 34: / Line 39: @@
 </div></b><br />
 <p style="color:black;text-indent:30px;">
-Part BBa_K811005 has been utilized by the 2012 Penn iGEM team to display a variety of different proteins. The red fluorescent protein mCherry has been successfully displayed on the surface of E. Coli, where it can produce fluorescence. We displayed 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/2012/8/82/MCherry-Here.gif" width="500" height="500"/></div></div>
+Part BBa_K811005 has been utilized by the 2012 Penn iGEM team to display a two completely different classes of proteins and we are currently using it to display a variety of other proteins. The red fluorescent protein mCherry has been successfully displayed on the surface of E. Coli, where it can produce fluorescence. We displayed 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>