Difference between revisions of "Part:BBa K2632008"
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We gave the surface display systerm from <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a> a new fuction though displaying RGD motif on the Lpp-OmpA in <i>E. coli</i>. RGD motif can specifically bind to αVβ3 a biomarker of cancer cells<sup>1</sup>. In addition, a rescent study successfully used <i>Salmonella</i> display RGD motif to target αVβ3-positive tumor cells<sup>2</sup>. We determine surface display site on the third loop of ompA though homology modeling (Figure 1). This part is under the control of <i>lac</i> promoter.
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<p>We gave the surface display system from  
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<br>
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a new function through displaying a RGD motif on
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<img src="https://static.igem.org/mediawiki/parts/4/4e/T--HZAU-China--Part-Improve-Figure1.jpeg" width="400px"/>
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                    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
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                    determine
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Figure 1. Homology modeling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.
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                    the surface display site on the third loop of Lpp-OmpA through homology modelling (<b>Figure 1</b>). This
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                    part is under the control of <i>lac</i> promoter. </p>
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Microscopy shows that E. coli expressed Lpp-OmpA-RGD induced by 0.1mM IPTG can bind to αVβ3-positive MDA-MB-231 cell line. Red arrow point location of bacteria (Figure 2). But can not bind to αVβ3-negative MCF7 cell line (Figure 3). We also use <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a> as a control. This strain can not 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 <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. (Click here to see method.)
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                <div style="width: 30%; margin: 0px auto">
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                    <img src="https://static.igem.org/mediawiki/2018/5/5f/T--HZAU-China--Improve1.png" width="100%" alt="">
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<img src="https://static.igem.org/mediawiki/parts/8/81/T--HZAU-China--Part-Improve-Figure2.png" width="1000px"/>
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                </div>
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                <p><b>Figure 1</b>. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.</p>
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Figure 2. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructive expressed RFP and contained contained <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. This improved part expresses RGD motif under control of <i>lac</i> promoter.  
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            </div>
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            <div id="float02">
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<img src="https://static.igem.org/mediawiki/parts/3/37/T--HZAU-China--Part-Improve-Figure3.png" width="1000px"/>
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                <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>
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                <div style="width: 90%; margin: 0px auto">
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                    <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width="100%" alt="">
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Figure 3. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructive expressed RFP and contained contained <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. This improved part expresses RGD motif under control of <i>lac</i> promoter.
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                </div>
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                <p><b>Figure 2</b>. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
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<img src="https://static.igem.org/mediawiki/parts/9/9c/T--HZAU-China--Part-Improve-Figure4.png" width="1000px"/>
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                    expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of
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                    <i>lac</i> promoter. </p>
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Figure 4. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructive expressed RFP and contained BBa_J36850.
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                <div style="width: 90%; margin: 0px auto">
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                    <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width="100%" alt="">
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<img src="https://static.igem.org/mediawiki/parts/0/0d/T--HZAU-China--Part-Improve-Figure5.png" width="1000px"/>
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                </div>
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                <p><b>Figure 3</b>. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructive expressed RFP
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Figure 5. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructive expressed RFP and contained BBa_J36850.
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                    and contained BBa_J36850. This improved part expressed RGD motif under the control of <i>lac</i> promoter.</p>
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                <div style="width: 90%; margin: 0px auto">
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                    <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width="100%" alt="">
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                </div>
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                <p><b>Figure 4</b>. αVβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constructively
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                    expressed RFP and contained BBa_J36850.</p>
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                <div style="width: 90%; margin: 0px auto">
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                    <img src="https://static.igem.org/mediawiki/2018/c/ce/T--HZAU-China--Improve5.png" width="100%" alt="">
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                </div>
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                <p><b>Figure 5</b>. αVβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constructively expressed RFP
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                    and contained BBa_J36850. </p>
  
  

Revision as of 18:37, 17 October 2018

Plac-Lpp-OmpA-RGD

We gave the surface display system from 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 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, adenocarcinoma1. We determine 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 by E. coli with 0.1mM IPTG can bind to αVβ3-positive MDA-MB-231 cell. Red arrow point the location of E. coli (Figure 2). But can not bind to αVβ3-negative MCF7 cell (Figure 3). We also use BBa_J36850 as a control. This strain can not 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 constructively 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 constructive 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 constructively expressed RFP and contained BBa_J36850.

Figure 5. αVβ3-negative MCF7 cell line was incubated with E. coli which constructively 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]