Difference between revisions of "Part:BBa K4768002"
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<partinfo>BBa_K4768002 parameters</partinfo>
 
<partinfo>BBa_K4768002 parameters</partinfo>
 
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
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            src="https://static.igem.wiki/teams/4768/wiki/parts/bba-k4768002.png">
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            <figcaption class="normal"><span class="titre-image"><i><b>Figure 1: Anti-HER2 nanobody part</b></i></span></figcaption>
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        </figure>
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<p>TXXXXXX.</p>
 
<p>TXXXXXX.</p>
  
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<h2>Construction</h2>
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<h2>Construction expression and purification</h2>
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<p>TXXXXXX.</p>
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            <figcaption class="normal"><span class="titre-image"><i><b>Figure 2: SDS-page migration  (15% acrylamid) on purification of periplasmic extract and Coomassie Blue revelation. Periplasmic extract (With IPTG or No IPTG), flowthrough (FT), wash (W), elution with 20 mM imidazole, 200 mM imidazole, 250 mM imidazole and 500 mM imidazole (respectively E1/20, E1/200, E1/250 and E1/ 500)
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<h2>Molecular Modeling</h2>
 
 
<p>TXXXXXX.</p>
 
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<h2>Characterisation</h2>
 
<h2>Characterisation</h2>
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<h3>Anchoring of liposomes decorated with Anti-HER2 nb on Caco-2 cells</h3>
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<p>400 nm fluorescent liposomes were prepared and coated with anti-HER2 nb, <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">see Protocol page</a>. Colorectal adenocarcinoma cells Caco-2, which are HER2-positive, were used to test the anchoring of liposomes by Anti-HER2 nb <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">see Protocol page</a>. Figure 1 shows a microscopy image of <b>adherent and non-adherent Caco-2 cells</b> observed in brightfield. For education purposes we appended on the image some features of living eukaryotic cells that can be distinguished with a standard optical microscope.</p>
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            <figcaption class="normal"><span class="titre-image"><i><b>Figure 3: Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode  with a 40X magnification. </b></i></span></figcaption>
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<p>Fluorescent liposomes were incubated on top of Caco-2 cells as described in our <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols" target="_blank">Protocol page</a>. Figure 3 shows the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancerous cells.</p>
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            <figcaption class="normal"><span class="titre-image"><i><b>Figure 3: Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode  with a 40X magnification. </b></i></span></figcaption>
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                                                    <iframe title="Optical imaging of Caco-2 cells and fluorescent liposomes functionalized with Anti-HER2 nb  after 1 hour incubation." width="800" height="700" src="https://video.igem.org/videos/embed/f09f1a4d-45fd-40fa-a395-01aed5fa4321?loop=1&amp;autoplay=1&amp;muted=1&amp;title=0&amp;warningTitle=0&amp;controlBar=0&amp;peertubeLink=0" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups"></iframe>
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                                            <p class="normal centered"><span class="titre-image"><i><b>Figure 4: Optical imaging of Caco-2 cells (brightfield) and 400-nm fluorescent liposomes (red fluorescence) functionalized with Anti-HER2 nb  after 1 hour incubation. This gif animation taken from a movie allows for categorizing liposomes as diffusing or immobile (anchored) during the lifespan of the movie.</b> </i></span></p>
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<h2>Conclusion and Perspectives</h2>
 
<h2>Conclusion and Perspectives</h2>

Revision as of 12:24, 8 October 2023


Anti-HER2 nanobody

Expression and purification of anti-Her2 nanobody for the anchoring to the liposome.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal SpeI site found at 166
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 166
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 518
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal SpeI site found at 166
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal SpeI site found at 166
    Illegal NgoMIV site found at 142
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

Figure 1: Anti-HER2 nanobody part

TXXXXXX.

Construction expression and purification

Figure 2: SDS-page migration (15% acrylamid) on purification of periplasmic extract and Coomassie Blue revelation. Periplasmic extract (With IPTG or No IPTG), flowthrough (FT), wash (W), elution with 20 mM imidazole, 200 mM imidazole, 250 mM imidazole and 500 mM imidazole (respectively E1/20, E1/200, E1/250 and E1/ 500)

TXXXXXX.

Characterisation

Anchoring of liposomes decorated with Anti-HER2 nb on Caco-2 cells

400 nm fluorescent liposomes were prepared and coated with anti-HER2 nb, see Protocol page. Colorectal adenocarcinoma cells Caco-2, which are HER2-positive, were used to test the anchoring of liposomes by Anti-HER2 nb see Protocol page. Figure 1 shows a microscopy image of adherent and non-adherent Caco-2 cells observed in brightfield. For education purposes we appended on the image some features of living eukaryotic cells that can be distinguished with a standard optical microscope.

Figure 3: Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode with a 40X magnification.

Fluorescent liposomes were incubated on top of Caco-2 cells as described in our Protocol page. Figure 3 shows the trajectory of liposomes over time. It is possible to differentiate between diffusing liposomes and anchored liposomes on cancerous cells.

Figure 3: Optical imaging of adherent and non-adherent Caco-2 cells. Cells were cultured in a petri dish and imaged with an inverted fluorescence microscope in the brightfield mode with a 40X magnification.

Figure 4: Optical imaging of Caco-2 cells (brightfield) and 400-nm fluorescent liposomes (red fluorescence) functionalized with Anti-HER2 nb after 1 hour incubation. This gif animation taken from a movie allows for categorizing liposomes as diffusing or immobile (anchored) during the lifespan of the movie.

Conclusion and Perspectives

TXXXXXX.

References

  1. article 1 xxxxxxxx
  2. article 2 xxxxxxx
    3.