Difference between revisions of "Part:BBa K1890002"

Revision as of 21:27, 19 October 2016

Silicatein gene, fused to transmembrane domain of OmpA, with strong RBS

Introduction

Silicatein, originating from the demosponge Tethya aurantia, catalyzes the formation of polysilicate. As described by Curnow et al., the silicatein gene was fused to the transmembrane domain of outer membrane protein A (OmpA), in order to display it at the surface of the cell [1][2]. The fusion of silicatein and OmpA is constructed according to Francisco et al., consisting of the transmembrane domain of OmpA together with the signaling peptide and the first nine N-terminal amino acids of lipoprotein (Lpp), both of which are native proteins from Escherichia coli (E. coli) [3]. The coding sequence in this BioBrick is set downstream of strong RBS BBa_B0034.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 192
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Silicatein is an enzyme natively found in demosponges and diatoms, where it catalyzes the condensation of silica to form the typical skeletal elements. Here, we use the enzyme to create a polysilicate layer around the host organism E. coli (Figure 1). The gene is fused to the transmembrane domain of OmpA in order to display the protein at the cell membrane. This biobrick was expressed under the control of an inducible promoter (Lac-promoter), to do so it was cloned in a backbone containing the promoter and all machinery necessary for it to work. This backbone was obtained from pBbS5a-RFP, a gift from Jay Keasling (Addgene plasmid #35283) [4].

**Figure 1:** Silicatein is able to convert monosilicate to polysilicate, allowing the cell to cover itself in polysilicate.

Characterization

This biobrick was expressed in the E. coli BL21 strain. Cells were grown overnight in selective LB. They were transferred to fresh medium and grown until in exponential phase. Then IPTG was added to induce expression. After a subsequent incubation of three hours, the medium was supplemented with silicic acid as substrate for silicatein. After another three hours, the silicate layer was considered to be formed [7]. A change in structure was observed for these cultures (Figure 2).

**Figure 2:** Structure of *E. coli* culture with polysilicate.

In order to characterize the formation of a polysilicate layer around E. coli, we performed multiple experiments.

Rhodamine 123 staining
Viability via a growth study
Scanning Electron Microscopy (SEM) imaging
Transmission Electron Microscopy (TEM) imaging
Analysis of physical properties with Atomic Force Microscopy (AFM)

Staining with Rhodamine 123

In this experiment we imaged the silicatein expressing cells with a fluorescence microscope, after treating them with a fluorescent dye. The fluorescent dye Rhodamine 123 (Sigma) has shown to bind specifically to polysilicate [5]. Cells were stained according to the protocol based on Li et al. and Müller et al. [5][6]. Rhodamine 123 was excited with a wavelength of 395 nm.

**Figure 3:** Widefield and fluorescence images of *E. coli* expressing OmpA-silicatein, treated with silicic acid (top) and without silicic acid (bottom). The cells were stained with Rhodamine 123 and excited at 395 nm. Of the widefield and fluorescence images an overlay was made to show the fraction of fluorescent cells.

From figure 3 we can see that the strain transformed with OmpA-silicatein clearly has a different output than the negative control. The fluorescence is only localized at the cells. From this we can conclude the Rhodamine 123 has stained the cells and therefore these cells are covered with a polysilicate layer.

Viability via a growth study

Since the silicatein expressing cells are to cover themselves in polysilicate, their nutrient supply might be limited by diffusion, which can eventually result in cell death. To investigate whether this is indeed the case, a growth study was performed (Figure 4). Cells were grown overnight in selective LB. They were transferred to fresh medium and grown until in exponential phase. Then IPTG was added to induce expression. After a subsequent incubation of three hours, the medium was supplemented with silicic acid as substrate for silicatein. During the following five hours samples were taken, of which many different dilutions were plated on selective LB plates. The day after colony forming units (cfu) were counted, and the 10^-6 dilution was the one that provided comparable results for all constructs tested, subsequently it was the dilution analysed. As a negative control, cells expressing this silicatein not supplemented with silicic acid were used.

**Figure 4:** Number of colony forming units (cfu) during incubation with silicic acid.

This figure shows that cells expressing this silicatein die after supplementing the medium with silicic acid, which suggests that either the polysilicate layer inhibits nutrient diffusion into the cell, or the silicic acid has a detrimental effect on growth. However, the Rhodamine 123 staining results show that silicatein works regardless of the state of the cells. The viability of the negative control culture decreases towards the end of the experiment due to the long incubation time.

SEM imaging

The polysilicate layer around the cells was prepared according to the polysilicate layer protocol. As a negative control, uninduces cultures (no IPTG) and cultures without substrate (no silicic acid) were used. The samples were fixed with gluteraldehyde and imaged with SEM. We used an FEI Niva Nano 450 SEM, under high vacuum.

**Figure 5:** SEM images of *E. coli* expressing OmpA-silicatein in the presence (A, B) or absence (C, D) of silicic acid.

First of all, since we know from the rhodamine staining experiment that the polysilicate layer is present, it does not seem to influence the cell shape. However, the cells that are expected to have a polysilicate layer appear to be somewhat fused together. According to Müller et al. [7], cells possessing a polysilicate layer appear to be fused by a viscous cover. This can, however, also be the result of limited imaging resolution. When using titanium oxide as a substrate [1] it is reported that large aggregates are visible by SEM. This was not observed in the current experiment suggesting that the polysilicate layer does not form aggregates or influence the shape of the cell, but forms a homogeneous layer around the cell.

TEM imaging

The experiment was performed using the E. coli BL21 strain with the plasmid containing OmpA-Silicatein. Two samples were made where the first sample was induced with IPTG but no silicic acid was added and a second sample which was both induced with IPTG and supplemented with silicic acid, so it would have a polysilicate layer. The samples were prepared by fixation using 1% polylysine on the surface of a Quantifoil carbon grid.

Both samples with and without silicic acid added were imaged using HAAFD-TEM and energy dispersive X-ray spectroscopy (figure 6). The cells are fixed at a Quantifoil carbon grid. In figure 6A and 6C the white structure is a cell laying on a hole in the grid. In each sample, we measured elemental composition of our sample including the silicon content (figure 6 B,D) the blue spots in these images indicate where silicon is detected. The grid itself already contains silicon but in the holes of the grid no silicon is present (figure 6 B,D). Therefore we measured the presence of silicon in bacteria laying on a hole on in the grid to make sure we do not have background silicon signal from the grid.

For the sample where no silicic acid is added (figure 6 A,B), we can see some silicon present at the position of the cell. However, there is a significant increase in silicon detected for the sample where silicic acid was added to the sample (figure 6D). This shows that silicon co-localizes with the cell which means there is indeed a polysilica layer formed by the bacteria.

Analysis of physical properties with AFM

Experiments were performed using E. coli BL21 strain transformed with the plasmid containing the OmpA-silicatein gene and induced with IPTG. Silicic acid was added to one sample to form the polysilicate layer around the cell. Another sample with no silicic acid added was used as a control. The cells were spun down and resuspended in MilliQ and fixated on a glass slide using 1% ABTES.

We have imaged two samples with AFM. The first sample is OmpA fused with silicatein with silicic acid added so that the cell can encapsulate itself with polysilicate (figure 7A,B). The second sample, that we used as a control, did not contain silicic acid (figure 7C,D). From both samples a height map (figure 7A,C) and the stiffness (figure 7B,D) was determined. Both samples were fixed on a glass slide in the same way. Due to a tip change is there a factor 10 difference between the stiffness of the glass slide of both samples.

Multiple cells (n=3) were imaged and the relative stiffness of the cell compared to the stiffness of the glass slide was determined (figure 7E). We found that cells covered with a layer of polysilicate have a stiffness of 0.43 compared to the glass slide and the cells without a layer of polysilicate have a stiffness of 0.16 compared to the glass slide. We can see that due to the encapsulation of the cells in polysilica the stiffness of the cells increases significantly.

References

[1] Curnow, P., Bessette, P. H., Kisailus, D., Murr, M. M., Daugherty, P. S., & Morse, D. E. (2005). Enzymatic synthesis of layered titanium phosphates at low temperature and neutral pH by cell-surface display of silicatein-a. Journal of the American Chemical Society, 127(45), 15749–15755.

[2] Curnow, P., Kisailus, D., & Morse, D. E. (2006). Biocatalytic synthesis of poly(L-lactide) by native and recombinant forms of the silicatein enzymes. Angewandte Chemie - International Edition, 45(4), 613–616.

[3] Francisco, J. a, Earhart, C. F., & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 89(April), 2713–2717.

[4] Lee, T. S., Krupa, R. A., Zhang, F., Hajimorad, M., Holtz, W. J., Prasad, N., … Keasling, J. D. (2011). BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Journal of Biological Engineering, 5, 12. http://doi.org/10.1186/1754-1611-5-12

[5] Li, C. W., Chu, S., & Lee, M. (1989). Characterizing the silica deposition vesicle of diatoms. Protoplasma, 151(2-3), 158–163.

[6] Müller, W. E. G., Rothenberger, M., Boreiko, A., Tremel, W., Reiber, A., & Schröder, H. C. (2005). Formation of siliceous spicules in the marine demosponge Suberites domuncula. Cell and Tissue Research, 321(2), 285–297.

[7] Müller, W. E. G., 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. http://doi.org/10.1016/j.biomaterials.2007.10.038

@@ Line 3: / Line 3: @@
 <h2>Introduction</h2>
-Silicatein, originating from the demosponge <i>Tethya aurantium</i>, catalyzes the formation of polysilicate.
+Silicatein, originating from the demosponge <i>Tethya aurantia</i>, catalyzes the formation of polysilicate.
-As described by Curnow <i>et al</i>, the silicatein gene was fused to the transmembrane domain of outer membrane
+As described by Curnow <i>et al.</i>, the silicatein gene was fused to the transmembrane domain of outer membrane
 protein A (OmpA), in order to display it at the surface of the cell [1][2].
-The fusion of silicatein and OmpA is constructed according to Francisco <i>et al</i>,
+The fusion of silicatein and OmpA is constructed according to Francisco <i>et al.</i>,
 consisting of the transmembrane domain of OmpA together with the signaling peptide and the first
-nine N-terminal amino acids of lipoprotein (Lpp), both of which are native proteins from <i>Escherichia coli</i> [3].
+nine N-terminal amino acids of lipoprotein (Lpp), both of which are native proteins from <i>Escherichia coli (E. coli)</i> [3].
 The coding sequence in this BioBrick is set downstream of strong RBS <partinfo>BBa_B0034</partinfo>.
@@ Line 20: / Line 20: @@
 The gene is fused to the transmembrane domain of OmpA in order to display the protein at the cell membrane.
 This biobrick was expressed under the control of an inducible promoter (Lac-promoter), to do so it was cloned in a backbone containing the promoter and all machinery necessary
-for it to work. This backbone was obtained from pBbS5a-RFP, a gift from Jay Keasling (Addgene plasmid # 35283) [4].
+for it to work. This backbone was obtained from pBbS5a-RFP, a gift from Jay Keasling (Addgene plasmid #35283) [4].
 <html>
@@ Line 26: / Line 26: @@
          <center><img src="https://static.igem.org/mediawiki/2016/4/43/T--TU_Delft--Silicate_layer.png">
              <figcaption>
-                 <b>Figure 1</b>: Silicatein is able to convert monosilicate to polysilicate, allowing the cell to cover itself in polysilicate.
+                 <b>Figure 1:</b> Silicatein is able to convert monosilicate to polysilicate, allowing the cell to cover itself in polysilicate.
              </figcaption></center>
      </figure>
@@ Line 32: / Line 32: @@
 <h2>Characterization</h2>
-This biobrick was expressed in <i>E. coli</i> BL21 strain. Cells were grown overnight in selective LB.
+This biobrick was expressed in the <i>E. coli</i> BL21 strain. Cells were grown overnight in selective LB.
 They were transferred to fresh medium and grown until in exponential phase.
 Then IPTG was added to induce expression. After a subsequent incubation of three hours,
@@ Line 43: / Line 43: @@
          <center><img src="https://static.igem.org/mediawiki/2016/7/76/T--TU_Delft--silicate_culture_structure.jpeg" width="50%">
              <figcaption>
-                 <b>Figure 2</b>: Structure of <i>E. coli</i> culture with polysilicate.
+                 <b>Figure 2:</b> Structure of <i>E. coli</i> culture with polysilicate.
              </figcaption></center>
      </figure>
@@ Line 50: / Line 50: @@
 In order to characterize the formation of a polysilicate layer around <i>E. coli</i>, we performed multiple experiments.
 <ul><li>Rhodamine 123 staining</li>
-<li> Growth study</li>
+<li> Viability via a growth study</li>
-<li> SEM imaging</li>
+<li> Scanning Electron Microscopy (SEM) imaging</li>
-<li> TEM imaging</li>
+<li> Transmission Electron Microscopy (TEM) imaging</li>
-<li> Analysis of physical properties with AFM</li>
+<li> Analysis of physical properties with Atomic Force Microscopy (AFM)</li>
 </ul>
@@ Line 66: / Line 66: @@
          <center><img src="https://static.igem.org/mediawiki/2016/8/8c/T--TU_Delft--silicatein92.png">
              <figcaption>
-                 <b>Figure 3</b>: Widefield and fluorescence images of <i>E. coli</i> expressing OmpA-silicatein, treated
+                 <b>Figure 3:</b> Widefield and fluorescence images of <i>E. coli</i> expressing OmpA-silicatein, treated
                  with silicic acid (top) and without silicic acid (bottom). The cells were stained with Rhodamine 123
                  and excited at 395 nm.
@@ Line 75: / Line 75: @@
 From figure 3 we can see that the strain transformed with OmpA-silicatein
-clearly has a different output from the negative control. The fluorescence is only
+clearly has a different output than the negative control. The fluorescence is only
 localized at the cells. From this we can conclude the Rhodamine 123 has stained the
 cells and therefore these cells are covered with a polysilicate layer.
-<h3>Viability</h3>
+<h3>Viability via a growth study</h3>
 Since the silicatein expressing cells are to cover themselves in polysilicate, their nutrient supply might be limited by diffusion, which can eventually result in cell death. To investigate whether this is indeed the case, a growth study was performed (Figure 4).
 Cells were grown overnight in selective LB. They were transferred to fresh medium and grown until in exponential phase. Then IPTG was added to induce expression. After a subsequent incubation of three hours, the medium was supplemented with silicic acid as substrate for silicatein. During the following five hours samples were taken, of which many different dilutions were plated on selective LB plates. The day after colony forming units (cfu) were counted, and the 10<sup>-6</sup> dilution was the one that provided comparable results for all constructs tested, subsequently it was the dilution analysed. As a negative control, cells expressing this silicatein not supplemented with silicic acid were used.
 <html>
      <figure>
-         <center><img src="https://static.igem.org/mediawiki/2016/d/dc/T--TU_Delft--viability_ompa.png">
+         <center><img src="https://static.igem.org/mediawiki/2016/a/ac/T--TU_Delft--viability_ompa_2.png">
              <figcaption>
-                 <b>Figure 4</b>: Number of colony forming units (cfu) during incubation with silicic acid.
+                 <b>Figure 4:</b> Number of colony forming units (cfu) during incubation with silicic acid.
              </figcaption></center>
      </figure>
 </html>
-This figure shows that cells expressing this silicatein die after supplementing the medium with silicic acid, which suggests that either the polysilicate layer inhibits nutrient diffusion into the cell, or the sodium silicate has a detrimental effect on growth. However, the Rhodamine 123 staining results show that silicatein works regardless of the state of the cells. The viability of the negative control culture decreases towards the end of the experiment due to the long incubation time.
+This figure shows that cells expressing this silicatein die after supplementing the medium with silicic acid, which suggests that either the polysilicate layer inhibits nutrient diffusion into the cell, or the silicic acid has a detrimental effect on growth. However, the Rhodamine 123 staining results show that silicatein works regardless of the state of the cells. The viability of the negative control culture decreases towards the end of the experiment due to the long incubation time.
 <h3>SEM imaging</h3>
-The polysilicate layer around the cells was prepared according to the polysilicate layer protocol. As a negative control, uninduces cultures (no IPTG) and cultures without substrate (no sodium silicate) were used. The samples were fixed with gluteraldehyde and imaged with SEM. We used an FEI Niva Nano 450 SEM, under high vacuum.
+The polysilicate layer around the cells was prepared according to the polysilicate layer protocol. As a negative control, uninduces cultures (no IPTG) and cultures without substrate (no silicic acid) were used. The samples were fixed with gluteraldehyde and imaged with SEM. We used an FEI Niva Nano 450 SEM, under high vacuum.
 <html>
      <figure>
           <center><img src="https://static.igem.org/mediawiki/2016/6/60/T--TU_Delft--SEM_imaging_silicatein_%281024x691%29.jpg" width="65%">
                <figcaption>
-                     <b>Figure 5</b>: SEM images of <i>E. coli</i> expressing OmpA-silicatein in the presence (A, B) or absence (C, D) of sodium silicate.
+                     <b>Figure 5:</b> SEM images of <i>E. coli</i> expressing OmpA-silicatein in the presence (A, B) or absence (C, D) of silicic acid.
                </figcaption></center>
       </figure>
 </html>
-First of all, since we know from the rhodamine staining experiment that the polysilicate layer is present, it does not seem to influence the cell shape. However, the cells that are expected to have a polysilicate layer appear to be somewhat fused together. According to Müller <i>et al</i> [7], cells possessing a polysilicate layer appear to be fused by a viscous cover. This can, however, also be the result of limited imaging resolution. When using titanium oxide as a substrate [1] it is reported that large aggregates are visible by SEM. This was not observed in the current experiment suggesting that the polysilicate layer does not form aggregated or influence the shape of the cell, but forms a homogeneous layer around the cell.
+First of all, since we know from the rhodamine staining experiment that the polysilicate layer is present, it does not seem to influence the cell shape. However, the cells that are expected to have a polysilicate layer appear to be somewhat fused together. According to Müller <i>et al.</i> [7], cells possessing a polysilicate layer appear to be fused by a viscous cover. This can, however, also be the result of limited imaging resolution. When using titanium oxide as a substrate [1] it is reported that large aggregates are visible by SEM. This was not observed in the current experiment suggesting that the polysilicate layer does not form aggregates or influence the shape of the cell, but forms a homogeneous layer around the cell.
 <h3>TEM imaging</h3>
-The experiment was performed using <i>E. coli</i> BL21 strain with the plasmid containing OmpA-Silicatein. Two samples were made where the first sample was induced with IPTG but no silicic acid was added and a second sample which was both induced with IPTG and supplemented with silicic acid, so it would have a polysilicate layer. The samples were prepared by fixation using 1% polylysine on the surface of a quantifoil carbon grid.
+The experiment was performed using the <i>E. coli</i> BL21 strain with the plasmid containing OmpA-Silicatein. Two samples were made where the first sample was induced with IPTG but no silicic acid was added and a second sample which was both induced with IPTG and supplemented with silicic acid, so it would have a polysilicate layer. The samples were prepared by fixation using 1% polylysine on the surface of a Quantifoil carbon grid.
-Both samples with and without silicic acid added were imaged using HAAFD-TEM and energy dispersive x-ray spectroscopy (figure 6). The cells are fixed at a Quantifoil carbon grid. In figure 6A and 6C the white structure is a cell laying on a hole in the grid. In each sample, we measured elemental composition of our sample including the silicon content (figure 6 B,D) the blue spots in these images indicate where silicon is detected. The grid itself already contains silicon but in the holes of the grid no silicon is present (figure 6 B,D). Therefore we measured the presence of silicon in bacteria laying on a hole on in the grid to make sure we do not have background silicon signal from the grid.
+Both samples with and without silicic acid added were imaged using HAAFD-TEM and energy dispersive X-ray spectroscopy (figure 6). The cells are fixed at a Quantifoil carbon grid. In figure 6A and 6C the white structure is a cell laying on a hole in the grid. In each sample, we measured elemental composition of our sample including the silicon content (figure 6 B,D) the blue spots in these images indicate where silicon is detected. The grid itself already contains silicon but in the holes of the grid no silicon is present (figure 6 B,D). Therefore we measured the presence of silicon in bacteria laying on a hole on in the grid to make sure we do not have background silicon signal from the grid.
 <html>
       <figure>
              <center><img src ="https://static.igem.org/mediawiki/2016/thumb/8/80/T--TU_Delft--TEM1.png/591px-T--TU_Delft--TEM1.png">
-                 <figcaption> <b>Figure 6</b>: (A,C) HAAFD image  and (B,D) EDX spectroscopy of silicon. (A,B). Image of the same cell containing OmpA-silicatein without silicic added to the sample (negative control). (C,D) Image of the same cell containing OmpA-Silicatein with silicic acid added to the sample.
+                 <figcaption> <b>Figure 6:</b> (A,C) HAAFD image  and (B,D) EDX spectroscopy of silicon. (A,B). Image of the same cell containing OmpA-silicatein without silicic added to the sample (negative control). (C,D) Image of the same cell containing OmpA-silicatein with silicic acid added to the sample.
                  </figcaption></center>
       </figure>
@@ Line 119: / Line 119: @@
 <h3>Analysis of physical properties with AFM</h3>
-Experiments were performed using <i>E. coli</i> BL21 strain transformed with the plasmid containing the OmpA-Silicatein gene and induced with IPTG. Silicic acid was added to one sample to form the polysilicate layer around the cell. Another sample with no silicic acid added was used as a control. The cells were spun down and resuspended in MilliQ and fixated on a glass slide using 1% ABTES.<br>
+Experiments were performed using <i>E. coli</i> BL21 strain transformed with the plasmid containing the OmpA-silicatein gene and induced with IPTG. Silicic acid was added to one sample to form the polysilicate layer around the cell. Another sample with no silicic acid added was used as a control. The cells were spun down and resuspended in MilliQ and fixated on a glass slide using 1% ABTES.<br>
 <br>
-We have imaged two samples with AFM. The first sample is OmpA fused with silicatein with silic acid added so that the cell can encapsulate  itself with biosilica (figure 7A-B). The second sample, that we used as a control, did not contain silicic acid (figure 7C-D).  From both samples a height map (figure 7A,C) and the stiffness (figure 7B,D) was determined. Both samples were fixed on a glass slide in the same way. Due to a tip change is there a factor 10 difference between the stiffness of the glass slide of both samples.
+We have imaged two samples with AFM. The first sample is OmpA fused with silicatein with silicic acid added so that the cell can encapsulate  itself with polysilicate (figure 7A,B). The second sample, that we used as a control, did not contain silicic acid (figure 7C,D).  From both samples a height map (figure 7A,C) and the stiffness (figure 7B,D) was determined. Both samples were fixed on a glass slide in the same way. Due to a tip change is there a factor 10 difference between the stiffness of the glass slide of both samples.
 <html>
     <figure>
         <center><img src = "https://static.igem.org/mediawiki/2016/thumb/e/e4/T--TU_Delft--AFM_Data1.png/800px-T--TU_Delft--AFM_Data1.png" alt= " " width="100%">
-             <figcaption> <b>Figure 7</b>: Pictures taken with AFM of (A-B) <i>E. coli</i> transformed with OmpA-fused silicatein with silicic acid added, (C-D) <i>E. coli</i> transformed with OmpA fused to silicatein without silic acid added. (A,C) are height maps of the cell, (B,D) are stiffness maps. (E) Relative stiffness of <i>E. coli</i> cells covered with and without polysilicate layer, compared to the stiffness of a glass slide measured with Peakforce QNM AFM.
+             <figcaption> <b>Figure 7:</b> Pictures taken with AFM of (A,B) <i>E. coli</i> transformed with OmpA-fused silicatein with silicic acid added, (C,D) <i>E. coli</i> transformed with OmpA fused to silicatein without silic acid added. (A,C) are height maps of the cell, (B,D) are stiffness maps. (E) Relative stiffness of <i>E. coli</i> cells covered with and without polysilicate layer, compared to the stiffness of a glass slide measured with Peakforce QNM AFM.
              </figcaption><center>
     </figure>