Difference between revisions of "Team:TU Delft/Proof"
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(Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890002" target="_blank">K1890002</a></b>) | (Part <b><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1890002" target="_blank">K1890002</a></b>) | ||
as shown by Rhodamine 123 staining of the polysilicate (Figure 1) and | as shown by Rhodamine 123 staining of the polysilicate (Figure 1) and | ||
| − | <a href="https://2016.igem.org/Team:TU_Delft/Project#silicatein" target="_blank"><b>other imaging experiments</b></a> | + | <a href="https://2016.igem.org/Team:TU_Delft/Project#silicatein" target="_blank"><b>other imaging experiments</b></a>. |
</p> | </p> | ||
<figure> | <figure> | ||
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</p> | </p> | ||
<figure> | <figure> | ||
| − | + | <center><img src="https://static.igem.org/mediawiki/2016/e/e7/T--TU_Delft--BolA_ind_widefield.png" width="60%"> | |
| − | + | <figcaption> | |
| − | + | <b>Figure 3</b>: Widefield images of <i>E. coli</i> BL21 transformed with a gene not altering the cell shape (<a href="http://parts.igem.org/Part:BBa_K1890002">K1890002</a>) (A) and with this part (B). | |
| − | + | </figcaption></center> | |
| − | + | </figure> | |
<p> | <p> | ||
When we express both the <i>BolA</i> gene as well as silicatein, we are able to construct round cells, coated in glass (Figure 4). | When we express both the <i>BolA</i> gene as well as silicatein, we are able to construct round cells, coated in glass (Figure 4). | ||
Revision as of 15:54, 18 October 2016
Functional proof of concept
Producing microlenses with bacteria
To produce biolenses we need our E. coli to perform two special activities: produce the a glass layer and change its shape from rod to round. To be able to obtain biological lenses we need a coating of polysilicate, biological glass, around the cell. This glass will give optical properties for the cell. E. coli is intrinsically not able to coat itself in polysilicate. However, upon transformation of the silicatein-α gene, originating from sponges, it is possible to coat the bacterium in a layer of polysilicate (Müller et al., 2008; Müller et al. 2003). Therefore, we are transforming E. coli with silicatein-α. We test silicatein from two different organisms expressed in three different ways, of which the most successful one was the construct consisting of silicatein from Tethya aurantia fused to the membrane protein OmpA (Part K1890002) as shown by Rhodamine 123 staining of the polysilicate (Figure 1) and other imaging experiments.
When making biological lenses, the shape of the lens is of crucial importance. E. coli is a rod-shaped organism, so it is not symmetrical along all axes. Shining light on the round parts of E. coli has a different effect on the focusing of light than shining light on the long sides (Figure 2). More information on this can be found on the modeling and project pages.
Biolenses with a spherical phenotype have an advantage over Biolenses with the rod-shaped E. coli phenotype, as for the round lenses, the orientation of the lens does not matter. The spherical cells we produced had an increased diameter compared to wildtype E. coli. The diameter of 1 µm that we observed matches the size of a photovoltaic cell, and is hard to produce using conventional techniques. Conventional microlenses are usually bigger. Therefore, our method of producing microlenses has an advantage over the conventional production, since we are able to produce far smaller lenses. Smaller lenses also means we can put more lenses on a surface, which increases the focusing effect.
In order to create spherical E. coli, we overexpress the BolA gene. BolA is a gene that controls the morphology of E. coli in the stress response (Santos et al. 1999). By overexpressing this gene, the rod-shaped E. coli cells will become round (Aldea et al., 1988). We express this gene both under a constitutive promoter (Part K1890031), as well as an inducible promoter (Part K1890030), the latter being our favorite due to the better result obtained (Figure 3).
When we express both the BolA gene as well as silicatein, we are able to construct round cells, coated in glass (Figure 4).
References
- Aldea, M., Hernandez-Chico, C., De La Campa, A., Kushner, S., & Vicente, M. (1988). Identification, cloning, and expression of bolA, an ftsZ-dependent morphogene of Escherichia coli. Journal of bacteriology, 170(11), 5169-5176.
- Müller, W. E. G. (2003). Silicon biomineralization.
- Müller, W. E., 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.
- Santos, J. M., Freire, P., Vicente, M., & Arraiano, C. M. (1999). The stationary‐phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Molecular microbiology, 32(4), 789-798.
