Part:BBa_K1890002
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 192
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
- Rhodamine 123 staining
- Growth study
- SEM imaging
- TEM imaging
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 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.
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