Difference between revisions of "Part:BBa K2632008"
Line 4: Line 4:
 
<html>
 
<html>
  
−
<p>We gave the surface display system from
+
<p>We gave the surface display system a new function through displaying a RGD motif on
−
a new function through displaying a RGD motif on
+
                     the Lpp-OmpA which contains a signal sequence, the N-terminal of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. In addition, lipoprotein performs the function of targeting to the outer membrane and OmpA constructs an anchor on the outer membrane. RGD motif can specifically bind to αvβ3, a biomarker of cancer cells such as melanoma, neuroblastoma, glioma and adenocarcinoma<sup>1</sup>. We determined the surface display site on the third loop of Lpp-OmpA through homology modelling (<b>Figure 1</b>). This part is under the control of <i>lac</i> promoter. </p>
−
                     the Lpp-OmpA which contains a signal sequence, the N-terminal of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. In addition, lipoprotein executes the function of targeting to the outer membrane and OmpA constructs an anchor on the outer membrane. RGD motif can specifically bind to αVβ3, a biomarker of cancer cells such as melanoma, neuroblastoma, glioma, adenocarcinoma<sup>1</sup>. We
+
−
                    determine
+
−
                    the surface display site on the third loop of Lpp-OmpA through homology modelling (<b>Figure 1</b>). This
+
−
                    part is under the control of <i>lac</i> promoter. </p>
+
 
                 <div style="width: 30%; margin: 0px auto">
 
                 <div style="width: 30%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/5/5f/T--HZAU-China--Improve1.png" width="100%" alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/5/5f/T--HZAU-China--Improve1.png" width="100%" alt="">
Line 16: Line 12:
 
             </div>
 
             </div>
 
             <div id="float02">
 
             <div id="float02">
−
                 <p>Microscopy shows that Lpp-OmpA-RGD overexpressed by <i>E. coli</i> with 0.1mM IPTG can bind to αVβ3-positive MDA-MB-231 cell. Red arrow point the location of <i>E. coli</i> (<b>Figure 2</b>). But can not bind to αVβ3-negative MCF7 cell (<b>Figure 3</b>). We also use BBa_J36850 as a control. This strain can not bind to Vβ3-positive MDA-MB-231 cell line (<b>Figure 4</b>) and αVβ3-negative MCF7 cell line (<b>Figure 5</b>). These results suggest that we successfully improve the part <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. </p>
+
                 <p>Microscopy shows that Lpp-OmpA-RGD overexpressed in <i>E. coli</i> induced by 0.1mM IPTG can bind to αvβ3-positive MDA-MB-231 cell (<b>Figure 2</b>), but cannot bind to αvβ3-negative MCF7 cell (<b>Figure 3</b>). We also use BBa_J36850 as a control. This strain cannot bind to Vβ3-positive MDA-MB-231 cell line (<b>Figure 4</b>) and αvβ3-negative MCF7 cell line (<b>Figure 5</b>). These results suggest that we successfully improve the part <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. </p>
 
                 <div style="width: 90%; margin: 0px auto">
 
                 <div style="width: 90%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width="100%" alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width="100%" alt="">
 
                 </div>
 
                 </div>
−
                 <p><b>Figure 2</b>. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
+
                 <p><b>Figure 2</b>. αvβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of
−
                    expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of
+
 
                     <i>lac</i> promoter. </p>
 
                     <i>lac</i> promoter. </p>
 
                 <div style="width: 90%; margin: 0px auto">
 
                 <div style="width: 90%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width="100%" alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width="100%" alt="">
 
                 </div>
 
                 </div>
−
                 <p><b>Figure 3</b>. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructive expressed RFP
+
                 <p><b>Figure 3</b>. αvβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of <i>lac</i> promoter.</p>
−
                    and contained BBa_J36850. This improved part expressed RGD motif under the control of <i>lac</i> promoter.</p>
+
 
                 <div style="width: 90%; margin: 0px auto">
 
                 <div style="width: 90%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width="100%" alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width="100%" alt="">
 
                 </div>
 
                 </div>
−
                 <p><b>Figure 4</b>. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
+
                 <p><b>Figure 4</b>. αvβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850.</p>
−
                    expressed RFP and contained BBa_J36850.</p>
+
 
                 <div style="width: 90%; margin: 0px auto">
 
                 <div style="width: 90%; margin: 0px auto">
 
                     <img src="https://static.igem.org/mediawiki/2018/c/ce/T--HZAU-China--Improve5.png" width="100%" alt="">
 
                     <img src="https://static.igem.org/mediawiki/2018/c/ce/T--HZAU-China--Improve5.png" width="100%" alt="">
 
                 </div>
 
                 </div>
−
                 <p><b>Figure 5</b>. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructively expressed RFP
+
                 <p><b>Figure 5</b>. αvβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850. </p>
−
                    and contained BBa_J36850. </p>
+
  
  

Revision as of 00:56, 18 October 2018

Plac-Lpp-OmpA-RGD

We gave the surface display system a new function through displaying a RGD motif on the Lpp-OmpA which contains a signal sequence, the N-terminal of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. In addition, lipoprotein performs the function of targeting to the outer membrane and OmpA constructs an anchor on the outer membrane. RGD motif can specifically bind to αvβ3, a biomarker of cancer cells such as melanoma, neuroblastoma, glioma and adenocarcinoma1. We determined the surface display site on the third loop of Lpp-OmpA through homology modelling (Figure 1). This part is under the control of lac promoter.

Figure 1. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.

Microscopy shows that Lpp-OmpA-RGD overexpressed in E. coli induced by 0.1mM IPTG can bind to αvβ3-positive MDA-MB-231 cell (Figure 2), but cannot bind to αvβ3-negative MCF7 cell (Figure 3). We also use BBa_J36850 as a control. This strain cannot bind to Vβ3-positive MDA-MB-231 cell line (Figure 4) and αvβ3-negative MCF7 cell line (Figure 5). These results suggest that we successfully improve the part BBa_J36850.

Figure 2. αvβ3-positive MDA-MB-231 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.

Figure 3. αvβ3-negative MCF7 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.

Figure 4. αvβ3-positive MDA-MB-231 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850.

Figure 5. αvβ3-negative MCF7 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850.

Reference

1. Park, S. H. et al. RGD peptide cell-surface display enhances the targeting and therapeutic efficacy of attenuated Salmonella-mediated cancer therapy. Theranostics 6, 1672–1682 (2016).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]