Difference between revisions of "Part:BBa K1890002"
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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 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 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 aggregated or influence the shape of the cell, but forms a homogeneous layer around the cell.
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<h3>TEM imaging</h3>
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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.
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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.
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            <center><img src ="https://static.igem.org/mediawiki/2016/thumb/8/80/T--TU_Delft--TEM1.png/591px-T--TU_Delft--TEM1.png"> 
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                <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.
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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.
  
 
<h2>References</h2>
 
<h2>References</h2>

Revision as of 11:16, 18 October 2016

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

Introduction

Silicatein, originating from the demosponge Tethya aurantium, 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 [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 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
  • Growth study
  • SEM imaging
  • TEM imaging
  • Analysis of physical properties with 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 from 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

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 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.

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 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.

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

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 aggregated or influence the shape of the cell, but forms a homogeneous layer around the cell.

TEM imaging

The experiment was performed using 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.

Figure 6: (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.
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.

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-?? 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