Team:TU Delft/Proof

iGEM TU Delft


Functional proof of concept

Producing microlenses with bacteria

To produce biolenses we need our E. coli to perform two special activities: produce 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.

Rhodamine staining
Figure 1, widefield and fluorescence images of OmpA-silicatein with silicic acid and OmpA-silicatein without silicic acid (negative control) at maximum excitation energy. Of the widefield and fluorescence images an overlay was made to show the fraction of fluorescent cells. The negative control causes overexposure of the camera, therefore the fluorescent image only gives one uniform signal.

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.

Modeling
Figure 2, Our models show that rod shaped lenses focus light in an orientation dependent way (A, B), but spherical lenses focus light in an independent way (C).

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. 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).

BolA widefield
Figure 3, Widefield images of E. coli BL21 transformed the OmpA, not altering the cell shape (A) and with BolA under an inducible promoter (B).

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. When we express both the BolA gene as well as silicatein gene, we are able to construct round cells, coated in glass (Figure 4).

BolA
Figure 4, SEM images of (A) E. coli BL21 without the BolA gene, (B) E. coli transformed with the BolA gene.

References

  1. 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.
  2. Müller, W. E. G. (2003). Silicon biomineralization.
  3. 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.
  4. 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.
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