Difference between revisions of "Part:BBa K1890002"

Revision as of 15:22, 6 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 combined with the 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.

Characterization

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

Widefield imaging of cells stained with Rhodamine 123
Growth study
SEM imaging
TEM imaging

Staining with Rhodamine 123

In this experiment we imaged the silicatein expressing cells with a wide field 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 was excited with a wavelength of 395 nm.

References

[1] 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.

[2] 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.

[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] 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.

@@ Line 3: / Line 3: @@
 <h2>Introduction</h2>
-Silicatein, originating from the demosponge <i>Tethya aurantia</i>, catalyzes the formation of polysilicate.
+Silicatein, originating from the demosponge <i>Tethya aurantium</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
 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>,
 consisting of the transmembrane domain of OmpA together with the signaling peptide and the first
-nine N-terminal amino acids of lipoprotein (Lpp) [3]. The coding sequence in this BioBrick is
+nine N-terminal amino acids of lipoprotein (Lpp), both of which are native proteins from <i>Escherichia coli</i> [3].
-combined with the strong RBS <partinfo>BBa_B0034</partinfo>.
+The coding sequence in this BioBrick is combined with the strong RBS <partinfo>BBa_B0034</partinfo>.
 <h2>Sequence and Features</h2>
 <partinfo>BBa_K1890002 SequenceAndFeatures</partinfo>
+<h2>Usage and Biology</h2>
+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>.
+<h2>Characterization</h2>
+In order to characterize the formation of a polysilicate layer around <i>E. coli</i>, we performed multiple experiments.
+<ul><li>Widefield imaging of cells stained with Rhodamine 123 </li>
+<li> Growth study</li>
+<li> SEM imaging</li>
+<li> TEM imaging</li></ul>
+<h3>Staining with Rhodamine 123</h3>
+In this experiment we imaged the silicatein expressing cells with a wide field 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 <i>et al.</i> and Müller <i>et al.</i> [4][5].
+Rhodamine was excited with a wavelength of 395 nm.
 <h2>References</h2>
@@ Line 21: / Line 39: @@
 [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] 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.
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