Difference between revisions of "Team:TU Delft/Proof"

 
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                     <h2 class="title-style-2">Producing microlenses with bacteria</h2>
 
                     <h2 class="title-style-2">Producing microlenses with bacteria</h2>
  
                     <p>To produce biolenses we need our <i>E. coli</i> to perform two special activities: produce the a glass layer and  
+
                     <p>To produce biolenses we need our <i>E. coli</i> to perform two special activities: produce a glass layer and change its shape from rod to spherical. To be able to obtain biological lenses we need a coating of polysilicate, biological glass, around the cell. This glass will give optical properties for the cell. <i>E. coli</i> is intrinsically not able to coat itself in polysilicate. However, upon transformation of the silicatein-α gene, originating from sponges, it is possible to coat the bacterium in a layer of polysilicate <a href="#references">(Müller <i>et al.</i>, 2008; Müller <i>et al.</i>, 2003)</a>. Therefore, we are transforming <i>E. coli</i> with silicatein-α. We test silicatein from two different organisms expressed in three different ways. The most successful construct consists of silicatein from <i>Tethya aurantia</i> fused to the membrane protein OmpA (Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890002" target="_blank">K1890002</a></b>) as shown by Rhodamine 123 staining of the polysilicate (Figure 1) and <a href="https://2016.igem.org/Team:TU_Delft/Project#silicatein" target="_blank"><b>other imaging experiments</b></a>.
                        change its shape from rod to round. To be able to obtain biological lenses we need a coating of polysilicate, biological glass,  
+
                        around the cell. This glass will give optical properties for the cell. <i>E. coli</i>  
+
                        is intrinsically not able to coat itself in polysilicate. However, upon transformation of the silicatein-α gene,  
+
                        originating from sponges, it is possible to coat the bacterium in a layer of polysilicate  
+
                        <a href="#references">(Müller et al., 2008; Müller et al. 2003)</a>. Therefore, we are transforming <i>E. coli</i> with
+
                        silicatein-α. We test silicatein from two different organisms expressed in three different ways, of which the
+
                        most successful one was the construct consisting of silicatein from <i>Tethya aurantia</i> fused to the membrane protein OmpA  
+
                        (Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890002" target="_blank">K1890002</a></b>)
+
                        as shown by Rhodamine 123 staining of the polysilicate (Figure 1) and  
+
                        <a href="https://2016.igem.org/Team:TU_Delft/Project#silicatein" target="_blank"><b>other imaging experiments</b></a>.
+
 
                     </p>
 
                     </p>
 
                     <div class = "row">
 
                     <div class = "row">
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                             <figure>
 
                             <figure>
 
                                 <img src="https://static.igem.org/mediawiki/2016/8/8c/T--TU_Delft--silicatein92.png" alt="Rhodamine staining">
 
                                 <img src="https://static.igem.org/mediawiki/2016/8/8c/T--TU_Delft--silicatein92.png" alt="Rhodamine staining">
                                 <figcaption><b>Figure 1,</b> widefield and fluorescence images of OmpA-silicatein with silicic acid and OmpA-silicatein without silicic acid (negative control) at maximum excitation energy. Of the widefield and fluorescence images an overlay was made to show the fraction of fluorescent cells. The negative control causes overexposure of the camera, therefore the fluorescent image only gives one uniform signal. </figcaption>
+
                                 <figcaption><b>Figure 1:</b> Widefield and fluorescence images of OmpA-silicatein with silicic acid and OmpA-silicatein without silicic acid (negative control) at maximum excitation energy. Of the widefield and fluorescence images an overlay was made to show the fraction of fluorescent cells. The negative control causes overexposure of the camera, therefore the fluorescent image only gives one uniform signal. </figcaption>
 
                             </figure>
 
                             </figure>
 
                         </div></div>
 
                         </div></div>
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                         so it is not symmetrical along all axes. Shining light on the round parts of <i>E. coli</i> has a different effect
 
                         so it is not symmetrical along all axes. Shining light on the round parts of <i>E. coli</i> has a different effect
 
                         on the focusing of light than shining light on the long sides (Figure 2). More information on this can be found  
 
                         on the focusing of light than shining light on the long sides (Figure 2). More information on this can be found  
                    on the <b><a href="https://2016.igem.org/Team:TU_Delft/Model" target="_blank">modeling</a></b> and  
+
                        on the <b><a href="https://2016.igem.org/Team:TU_Delft/Model#lenses" target="_blank">modeling</a></b> and  
                    <a href="https://2016.igem.org/Team:TU_Delft/Project#silicatein" target="_blank"><b>project</b></a> pages.
+
                        <a href="https://2016.igem.org/Team:TU_Delft/Project#silicatein" target="_blank"><b>project</b></a> pages.
 
                     <div class = "row">
 
                     <div class = "row">
 
                         <div class="col-md-10 col-md-offset-1 col-sm-12">
 
                         <div class="col-md-10 col-md-offset-1 col-sm-12">
 
                             <figure>
 
                             <figure>
 
                                 <img src="https://static.igem.org/mediawiki/2016/b/b8/T--TU_Delft--light_focussing_model.png" alt="Modeling"">
 
                                 <img src="https://static.igem.org/mediawiki/2016/b/b8/T--TU_Delft--light_focussing_model.png" alt="Modeling"">
                                 <figcaption><b>Figure 2,</b> Our models show that rod shaped lenses focus light in an orientation dependent way (A, B),
+
                                 <figcaption><b>Figure 2:</b> Our models show that rod shaped lenses focus light in an orientation dependent way (A, B),
                            but spherical lenses focus light in an independent way (C).</figcaption>
+
                                    but spherical lenses focus light in an orientation independent way (C).</figcaption>
 
                             </figure>
 
                             </figure>
 
                         </div></div>     
 
                         </div></div>     
                 
 
                    <p>  Biolenses with a spherical phenotype have an advantage over Biolenses with the rod-shaped <i>E. coli</i> phenotype,
 
                        as for the round lenses, the orientation of the lens does not matter. The spherical cells we produced had an increased
 
                        diameter compared to wildtype <i>E. coli</i>. The diameter of 1 µm that we observed matches the size of a photovoltaic
 
                        cell, and is hard to produce using conventional techniques. Conventional microlenses are usually bigger. Therefore,
 
                        <strong>our method of producing microlenses has an advantage over the conventional production</strong>, since we
 
                        are able to produce far smaller lenses. Smaller lenses also means we can put more lenses on a surface,
 
                        which increases the focusing effect.
 
                    </p>
 
  
                     <p>In order to create spherical <i>E. coli</i>, we overexpress the <i>BolA</i> gene.  
+
                     <p> Biolenses with a spherical phenotype have an advantage over biolenses with the rod-shaped <i>E. coli</i> phenotype, as for the round lenses, orientation does not matter. In order to create spherical <i>E. coli</i>, we overexpress the <i>BolA</i> gene. <i>BolA</i> is a gene that controls the morphology of <i>E. coli</i> in the stress response <a href="#references">(Santos <i>et al.</i>, 1999)</a>. By overexpressing this gene, the rod-shaped <i>E. coli</i> cells will become spherical <a href="#references">(Aldea <i>et al.</i>, 1988)</a>. We express this gene both under a constitutive promoter (Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890031" target="_blank">K1890031</a></b>), as well as an inducible promoter (Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890030" target="_blank">K1890030</a></b>), the latter being our favorite due to the better result obtained (Figure 3).</p>
                        <i>BolA</i> is a gene that controls the morphology of <i>E. coli</i> in the stress  
+
                        response <a href="#references">(Santos <i>et al.</i> 1999)</a>.  
+
                        By overexpressing this gene, the rod-shaped <i>E. coli</i> cells will become  
+
                        round <a href="#references">(Aldea <i>et al.</i>, 1988)</a>. We express this gene both under a  
+
                        constitutive promoter (Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890031" target="_blank">K1890031</a></b>), as well as an inducible  
+
                        promoter (Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890030" target="_blank">K1890030</a></b>), the latter being our favorite
+
                        due to the better result obtained (Figure 3).
+
                    </p>
+
 
                     <div class = "row">
 
                     <div class = "row">
 
                         <div class="col-md-10 col-md-offset-1 col-sm-12">
 
                         <div class="col-md-10 col-md-offset-1 col-sm-12">
                             <figure>
+
                             <center><figure>
                                <img src="https://static.igem.org/mediawiki/2016/e/e7/T--TU_Delft--BolA_ind_widefield.png" alt="BolA widefield">
+
                                    <img src="https://static.igem.org/mediawiki/2016/e/e7/T--TU_Delft--BolA_ind_widefield.png" alt="BolA widefield">
                                <figcaption><b>Figure 3,</b> Widefield images of <i>E. coli</i> BL21 transformed with a gene not altering the cell shape (<a href="http://parts.igem.org/Part:BBa_K1890002">K1890002</a>) (A) and with this part (B).</figcaption>
+
                                    </center>
                            </figure>
+
                                    <figcaption><b>Figure 3:</b> Widefield images of <i>E. coli</i> BL21 transformed the OmpA-silicatein construct not altering the cell shape (A). When the cells are also transformed with the BolA under an inducible promoter the cells become spherical (B).</figcaption>
 +
                                </figure>
 
                         </div></div>
 
                         </div></div>
 
                     <p>
 
                     <p>
                         When we express both the <i>BolA</i> gene as well as silicatein, we are able to construct round cells, coated in glass (Figure 4).
+
                         The spherical cells we produced had an increased volume compared to wildtype <i>E. coli</i>. The diameter of 1 µm that we observed matches the size of a photovoltaic cell (the smallest unit of a solar panel)<a href="#references">(Yang, Shtein, & Forrest, 2005)</a>, and this size is hard to produce using conventional techniques. Conventional microlenses are usually bigger. Therefore, <strong>our method of producing microlenses has an advantage over the conventional production</strong>, since we are able to produce far smaller lenses. Using smaller lenses also means we are able to put more lenses on a surface, which increases the focusing effect. When we express both the <i>BolA</i> gene as well as silicatein gene, we are able to construct round cells, coated in glass (Figure 4).  
 
                     </p>
 
                     </p>
 
                     <div class = "row">
 
                     <div class = "row">
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                             <figure>
 
                             <figure>
 
                                 <img src="https://static.igem.org/mediawiki/2016/c/c2/T--TU_Delft--BolA_SEM.png" alt="BolA">
 
                                 <img src="https://static.igem.org/mediawiki/2016/c/c2/T--TU_Delft--BolA_SEM.png" alt="BolA">
                                 <figcaption><b> Figure 4, </b> SEM images of <b>(A)</b> <i>E. coli</i> BL21 without the <i>BolA</i> gene, <b>(B)</b> <i>E. coli</i> transformed with the <i>BolA</i> gene. </figcaption>
+
                                 <figcaption><b> Figure 4: </b> SEM images of <i>E. coli</i> BL21 without the <i>BolA</i> gene covered in polysilicate (A) and <i>E. coli</i> BL21 transformed with the <i>BolA</i> gene covered in polysilicate (B). </figcaption>
 
                             </figure>
 
                             </figure>
 
                         </div></div>
 
                         </div></div>
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                 <li>Müller, W. E., Engel, S., Wang, X., Wolf, S. E., Tremel, W., Thakur, N. L., Schröder, H. C. (2008). Bioencapsulation of living bacteria (Escherichia coli) with poly (silicate) after transformation with silicatein-α gene. Biomaterials, 29(7), 771-779. </li>
 
                 <li>Müller, W. E., Engel, S., Wang, X., Wolf, S. E., Tremel, W., Thakur, N. L., Schröder, H. C. (2008). Bioencapsulation of living bacteria (Escherichia coli) with poly (silicate) after transformation with silicatein-α gene. Biomaterials, 29(7), 771-779. </li>
 
                 <li>Santos, J. M., Freire, P., Vicente, M., & Arraiano, C. M. (1999). The stationary‐phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Molecular microbiology, 32(4), 789-798. </li>
 
                 <li>Santos, J. M., Freire, P., Vicente, M., & Arraiano, C. M. (1999). The stationary‐phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Molecular microbiology, 32(4), 789-798. </li>
 
+
                <li>Yang, F., Shtein, M., & Forrest, S. R. (2005). Controlled growth of a molecular bulk heterojunction photovoltaic cell. Nature materials, 4(1), 37-41. </li> 
 +
             
 
             </ol>
 
             </ol>
 
         </div>
 
         </div>

Latest revision as of 23:38, 19 October 2016

iGEM TU Delft


Functional proof of concept

Producing microlenses with bacteria

To produce biolenses we need our E. coli to perform two special activities: produce a glass layer and change its shape from rod to spherical. To be able to obtain biological lenses we need a coating of polysilicate, biological glass, around the cell. This glass will give optical properties for the cell. E. coli is intrinsically not able to coat itself in polysilicate. However, upon transformation of the silicatein-α gene, originating from sponges, it is possible to coat the bacterium in a layer of polysilicate (Müller et al., 2008; Müller et al., 2003). Therefore, we are transforming E. coli with silicatein-α. We test silicatein from two different organisms expressed in three different ways. The most successful construct consists of silicatein from Tethya aurantia fused to the membrane protein OmpA (Part K1890002) as shown by Rhodamine 123 staining of the polysilicate (Figure 1) and other imaging experiments.

Rhodamine staining
Figure 1: Widefield and fluorescence images of OmpA-silicatein with silicic acid and OmpA-silicatein without silicic acid (negative control) at maximum excitation energy. Of the widefield and fluorescence images an overlay was made to show the fraction of fluorescent cells. The negative control causes overexposure of the camera, therefore the fluorescent image only gives one uniform signal.

When making biological lenses, the shape of the lens is of crucial importance. E. coli is a rod-shaped organism, so it is not symmetrical along all axes. Shining light on the round parts of E. coli has a different effect on the focusing of light than shining light on the long sides (Figure 2). More information on this can be found on the modeling and project pages.

Modeling
Figure 2: Our models show that rod shaped lenses focus light in an orientation dependent way (A, B), but spherical lenses focus light in an orientation independent way (C).

Biolenses with a spherical phenotype have an advantage over biolenses with the rod-shaped E. coli phenotype, as for the round lenses, orientation does not matter. In order to create spherical E. coli, we overexpress the BolA gene. BolA is a gene that controls the morphology of E. coli in the stress response (Santos et al., 1999). By overexpressing this gene, the rod-shaped E. coli cells will become spherical (Aldea et al., 1988). We express this gene both under a constitutive promoter (Part K1890031), as well as an inducible promoter (Part K1890030), the latter being our favorite due to the better result obtained (Figure 3).

BolA widefield
Figure 3: Widefield images of E. coli BL21 transformed the OmpA-silicatein construct not altering the cell shape (A). When the cells are also transformed with the BolA under an inducible promoter the cells become spherical (B).

The spherical cells we produced had an increased volume compared to wildtype E. coli. The diameter of 1 µm that we observed matches the size of a photovoltaic cell (the smallest unit of a solar panel)(Yang, Shtein, & Forrest, 2005), and this size is hard to produce using conventional techniques. Conventional microlenses are usually bigger. Therefore, our method of producing microlenses has an advantage over the conventional production, since we are able to produce far smaller lenses. Using smaller lenses also means we are able to put more lenses on a surface, which increases the focusing effect. When we express both the BolA gene as well as silicatein gene, we are able to construct round cells, coated in glass (Figure 4).

BolA
Figure 4: SEM images of E. coli BL21 without the BolA gene covered in polysilicate (A) and E. coli BL21 transformed with the BolA gene covered in polysilicate (B).
  1. Aldea, M., Hernandez-Chico, C., De La Campa, A., Kushner, S., & Vicente, M. (1988). Identification, cloning, and expression of bolA, an ftsZ-dependent morphogene of Escherichia coli. Journal of bacteriology, 170(11), 5169-5176.
  2. Müller, W. E. G. (2003). Silicon biomineralization.
  3. Müller, W. E., Engel, S., Wang, X., Wolf, S. E., Tremel, W., Thakur, N. L., Schröder, H. C. (2008). Bioencapsulation of living bacteria (Escherichia coli) with poly (silicate) after transformation with silicatein-α gene. Biomaterials, 29(7), 771-779.
  4. Santos, J. M., Freire, P., Vicente, M., & Arraiano, C. M. (1999). The stationary‐phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Molecular microbiology, 32(4), 789-798.
  5. Yang, F., Shtein, M., & Forrest, S. R. (2005). Controlled growth of a molecular bulk heterojunction photovoltaic cell. Nature materials, 4(1), 37-41.