Difference between revisions of "Part:BBa K2632008"
StephanieHWX (Talk | contribs) |
|||
| (3 intermediate revisions by 2 users not shown) | |||
| Line 4: | Line 4: | ||
<html> | <html> | ||
| − | We gave the surface display | + | <p>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 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> |
| − | <img src="https://static.igem.org/mediawiki/ | + | <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=""> |
| − | Figure 1. | + | </div> |
| − | <br> | + | <p><b>Figure 1</b>. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.</p><br> |
| − | Microscopy shows that | + | </div> |
| − | < | + | <div id="float02"> |
| − | <img src="https://static.igem.org/mediawiki/ | + | <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 improved the part <a href="https://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. </p> |
| − | < | + | <div style="width: 90%; margin: 0px auto"> |
| − | Figure 2. | + | <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width="100%" alt=""> |
| − | < | + | </div> |
| − | <img src="https://static.igem.org/mediawiki/ | + | <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 |
| − | < | + | <i>lac</i> promoter. </p> |
| − | + | <div style="width: 90%; margin: 0px auto"> | |
| − | Figure 3. | + | <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width="100%" alt=""> |
| − | < | + | </div> |
| − | <img src="https://static.igem.org/mediawiki/ | + | <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> |
| − | < | + | <div style="width: 90%; margin: 0px auto"> |
| − | Figure 4. | + | <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width="100%" alt=""> |
| − | + | </div> | |
| − | <img src="https://static.igem.org/mediawiki/ | + | <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> |
| − | < | + | <div style="width: 90%; margin: 0px auto"> |
| − | Figure 5. | + | <img src="https://static.igem.org/mediawiki/2018/c/ce/T--HZAU-China--Improve5.png" width="100%" alt=""> |
| + | </div> | ||
| + | <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> | ||
Latest revision as of 03:44, 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 improved 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]