<b><div class="name" align="center">Generalized Surface Display System
-
</div></b><br />
-
<p style="color:black;text-indent:30px;">
-
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>
-
-
<p style="color:black;text-indent:30px;">
-
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>
<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>
Ice nucleation protein (INP) is a protein found in Xanthomonas campestris 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.
Experience
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).
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.
"
