Difference between revisions of "Part:BBa K1890001"

 
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<partinfo>BBa_K1890001 short</partinfo>
 
<partinfo>BBa_K1890001 short</partinfo>
  
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Silicatein, originating from the demosponge Suberites domuncula, catalyzes the formation of polysilicate. The gene is fused to the transmembrane domain of outer membrane protein A (OmpA), a native protein from E. coli, together with Lpp signaling peptide.
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<h2>Introduction</h2>
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Silicatein, originating from the demosponge <i>Suberites domuncula</i>, catalyzes the formation of polysilicate. This biobrick contains the short version of the silicatein gene, according to Müller <i>et al</i> [1][2]. The gene is fused to the transmembrane domain of ice nucleation protein (INP) from <i>Pseudomonas syringae</i> [3]. The coding sequence in this BioBrick is set downstream of the strong RBS <partinfo>BBa_B0034</partinfo>.
  
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<h2>Sequence and Features</h2>
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1890001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1890001 SequenceAndFeatures</partinfo>
  
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<h2>Usage and Biology</h2>
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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 <i>E. coli</i> (Figure 1).
 +
The gene is fused to the transmembrane domain of INP 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) [6].
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<html>
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    <figure>
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        <center><img src="https://static.igem.org/mediawiki/2016/4/43/T--TU_Delft--Silicate_layer.png">
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            <figcaption>
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                <b>Figure 1</b>: Silicatein is able to convert monosilicate to polysilicate, allowing the cell to cover itself in polysilicate.
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            </figcaption></center>
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    </figure>
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</html>
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<h2>Characterization</h2>
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In order to characterize the formation of a polysilicate layer around <i>E. coli</i>, we performed the following experiments.
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<ul><li>Rhodamine 123 staining</li>
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    <li> Growth study</li></ul>
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<h3>Staining with Rhodamine 123</h3>
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In this experiment we imaged the silicatein expressing cells with a fluorescence microscope, after treating them with a fluorescent dye.
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The fluorescent dye Rhodamine 123 (Sigma) has shown to bind specifically to polysilicate [4].
 +
Cells were stained according to the protocol based on Li <i>et al.</i> and Müller <i>et al.</i> [4][5].
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Rhodamine 123 was excited with a wavelength of 488 nm.
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<html>
 +
    <figure>
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        <center><img src="https://static.igem.org/mediawiki/2016/8/89/Silicatein_microscopy_3_INP.png">
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            <figcaption>
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                <b>Figure 2</b>: Widefield and fluorescence images of <i>E. coli</i> expressing INP-silicatein, treated
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                with silicic acid (top) and without silicic acid (bottom). The cells were stained with Rhodamine 123
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                and excited at 488 nm.
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                Of the widefield and fluorescence images an overlay was made to show the fraction of fluorescent cells.
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            </figcaption></center>
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    </figure>
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</html>
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We cannot distinguish a clear difference between silicatein and the negative control. The entire medium is fluorescent, which causes overexposure of the camera at high excitation intensity. This might mean that the Rhodamine 123 is not specifically located at the cell walls, but still dissolved in the medium. Wedo see some fluorescence localized at the cells, but the diference between the fluorescence of the medium and the cells is much smaller than we observed for OmpA-Silicatein (<partinfo>K1890002</partinfo>). Therefore, we cannot conclude that the strains transformed with the INP-silicatein plasmid are able to synthesize a polysilicate layer around the cell.
 +
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<h3>Viability</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 3).
 +
Cells were grown overnight in selective LB. They were transfered 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 another type of silicatein <partinfo>K1890002</partinfo>, not supplemented with silicic acid were used.
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<html>
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    <figure>
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        <center><img src="https://static.igem.org/mediawiki/2016/f/f3/T--TU_Delft--viability_inp.png">
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            <figcaption>
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                <b>Figure 3</b>: Number of colony forming units (cfu) during incubation with silicic acid.
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            </figcaption></center>
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    </figure>
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</html>
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This figure suggests that either  the sodium silicate has a detrimental effect on growth or that, in case there is a polysilicate layer (which we were not able to confirm with Rhodamine 123 staining), it inhibits nutrient diffusion into the cell.
 +
 +
<b>USAFA iGEM Team contribution:</b>
 +
The 2024 USAFA iGEM Team has successfully been able to express INP-silicatein in E. coli and have been able to produce viable colonies. We have found that adding the orthosilicate after log phase growth allows the bacteria to grow to sufficient density. We only add the orthosilicate at the end when we are ready for biosilicification to happen. 4mM orthosilicate was used and we were able to get silica precipitation from the E. coli expressing INP-sil. See part BBa_K5199004 for the updated INP-silicatein coding sequence with a new truncated and codon-optimized nucleotide sequence (K5199003). The new INP-sil composite part (K5199004) has data and successfully shows SiO2 precipitation in biocement using E. coli INP-sil.
 +
 +
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<h2>References</h2>
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[1] Müller, W. E. G., Engel, S., Wang, X., Wolf, S. E., Tremel, W., Thakur, N. L., … Schröder, H. C. (2008). Bioencapsulation of living bacteria (<i>Escherichia coli</i>) with poly(silicate) after transformation with silicatein-α gene. Biomaterials, 29(7), 771–779.
 +
 +
[2] Müller, W. E. G. (2003). Silicon biomineralization.
 +
 +
[3] Kim, E., & Yoo, S. (1998). Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein, 12(3), 197–201.
 +
 +
[4] Li, C. W., Chu, S., & Lee, M. (1989). Characterizing the silica deposition vesicle of diatoms. Protoplasma, 151(2-3), 158–163.
 +
 +
[5] 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.
 +
 +
[6] 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
  
 
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Latest revision as of 23:12, 30 September 2024

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

Introduction

Silicatein, originating from the demosponge Suberites domuncula, catalyzes the formation of polysilicate. This biobrick contains the short version of the silicatein gene, according to Müller et al [1][2]. The gene is fused to the transmembrane domain of ice nucleation protein (INP) from Pseudomonas syringae [3]. The coding sequence in this BioBrick is set downstream of the strong RBS BBa_B0034.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 98
    Illegal NgoMIV site found at 431
  • 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 INP 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) [6].

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

Characterization

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

  • Rhodamine 123 staining
  • Growth study

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 [4]. Cells were stained according to the protocol based on Li et al. and Müller et al. [4][5]. Rhodamine 123 was excited with a wavelength of 488 nm.

Figure 2: Widefield and fluorescence images of E. coli expressing INP-silicatein, treated with silicic acid (top) and without silicic acid (bottom). The cells were stained with Rhodamine 123 and excited at 488 nm. Of the widefield and fluorescence images an overlay was made to show the fraction of fluorescent cells.

We cannot distinguish a clear difference between silicatein and the negative control. The entire medium is fluorescent, which causes overexposure of the camera at high excitation intensity. This might mean that the Rhodamine 123 is not specifically located at the cell walls, but still dissolved in the medium. Wedo see some fluorescence localized at the cells, but the diference between the fluorescence of the medium and the cells is much smaller than we observed for OmpA-Silicatein (BBa_K1890002). Therefore, we cannot conclude that the strains transformed with the INP-silicatein plasmid are able to synthesize a polysilicate layer around the cell.

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 3). Cells were grown overnight in selective LB. They were transfered 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 another type of silicatein BBa_K1890002, not supplemented with silicic acid were used.

Figure 3: Number of colony forming units (cfu) during incubation with silicic acid.

This figure suggests that either the sodium silicate has a detrimental effect on growth or that, in case there is a polysilicate layer (which we were not able to confirm with Rhodamine 123 staining), it inhibits nutrient diffusion into the cell.

USAFA iGEM Team contribution: The 2024 USAFA iGEM Team has successfully been able to express INP-silicatein in E. coli and have been able to produce viable colonies. We have found that adding the orthosilicate after log phase growth allows the bacteria to grow to sufficient density. We only add the orthosilicate at the end when we are ready for biosilicification to happen. 4mM orthosilicate was used and we were able to get silica precipitation from the E. coli expressing INP-sil. See part BBa_K5199004 for the updated INP-silicatein coding sequence with a new truncated and codon-optimized nucleotide sequence (K5199003). The new INP-sil composite part (K5199004) has data and successfully shows SiO2 precipitation in biocement using E. coli INP-sil.


References

[1] 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.

[2] Müller, W. E. G. (2003). Silicon biomineralization.

[3] Kim, E., & Yoo, S. (1998). Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein, 12(3), 197–201.

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

[5] 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.

[6] 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