Team:TU Delft/Composite Part

iGEM TU Delft

Best composite part

OmpA-silicatein

Producing microlenses with bacteria

Abstract

The essential activity that our Escherichia coli needs to perform to create biolenses is surround itself by a glass layer. We have achieved this thanks to part K1890002, which we have characterized in several experiments and we have proven that it works as expected. This part consists of the silicatein enzyme from Tethya aurantia fused to the Outer membrane protein A (OmpA) from E. coli.

Design

The glass layer is produced by silicatein-α, which is original from sponges and catalyzes the conversion of silicic acid to polysilicate from monomeric silicic acid (Müller et al., 2005; Müller et al., 2008). To make sure that the cell is coated by polysilicate we engineered a fusion protein combining the silicatein-α gene from T. aurantia to OmpA from E. coli, we have called this new BioBrick OmpA-silicatein (Part K1890002). Once the fusion protein was designed the sequence was codon-optimized for expression in E. coli.

We expressed this construct under the control of an inducible promoter (Lac-promoter), which was present in the plasmid backbone we used, together with the LacI gene. This backbone was obtained from pBbA5c-RFP, a gift from Jay Keasling (Addgene plasmid # 35281) (Lee et al., 2011). Upon transformation of this plasmid in BL21 E. coli cells and after induction with IPTG and supplementing the growth medium with silicic acid (the substrate for silicatein to produce the polysilicate layer), our cells were covered by a polysilicate layer as shown by Rhodamine 123 staining, TEM and AFM.

Rhodamine 123 staining

Rhodamine 123 is a fluorescent stain that binds to polysilicate. thus, by staining our cells with it and imaging them with fluorescence microscopy we are be able to determine whether they are covered by polysilicate (Figure 1).

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.

In figure 1 we can see that the strain transformed with OmpA-silicatein clearly has a different output from the negative control. The fluorescence of this sample is only localized at the cells. This might mean that the Rhodamine 123 has stained these cells and therefore the OmpA-silicatein cells could have the polysilicate layer around their membranes.

Imaging of silicatein-expressing cells and their elemental composition using TEM

Our cells expressing the OmpA-silicatein construct were imaged using HAAFD-TEM and energy dispersive x-ray spectroscopy (figure 2). For this, a negative control of induced cells not supplemented with silicic acid was used. In each sample, we measured elemental composition of our sample including the silicon content (figure 2 B,D) the blue spots in these images indicate where silicon is detected. Since the grid itself also contains silicon, we only measured the presence of silicon in bacteria laying on a hole on in the grid to make sure we do not have background silicon signal from the grid, which cannot be distinguished from the actual signal.

Figure 2: (A,C) HAAFD image and (B,D) EDX spectroscopy of silicon. (A,B). Image of the same cell containing OmpA-silicatein without silicic added to the sample (negative control). (C,D) Image of the same cell containing OmpA-Silicatein with silicic acid added to the sample.

For the sample where no silicic acid is added (figure 2 A,B), we can see some silicon present at the position of the cell. However, there is a significant increase in silicon detected for the sample where silicic acid was added to the sample (figure 2D). This shows that silicon co-localizes with the cell which means there is indeed a polysilicate layer formed by the bacteria.

Analysis of physical properties of polysilicate covered cells using AFM

We have imaged the two samples from TEM also with AFM. From both samples a height map (figure 3A, C) and the stiffness (figure 3B, D) was determined. Both samples were fixed on a glass slide in the same way. Due to a tip change is there a factor 10 difference between the measured stiffness of the glass slide of both samples.

Figure 3: Pictures taken with AFM of (A-B) E. coli transformed with OmpA-silicatein with silicic acid added, (C-D) E. coli transformed with OmpA-silicatein without silic acid added. (A,C) are height maps of the cell, (B,D) are stiffness maps. (E) Relative stiffness of E. coli cells covered with and without polysilicate layer, compared to the stiffness of a glass slide measured with Peakforce QNM AFM.

Multiple cells (n=3) were imaged and the relative stiffness of the cell compared to the stiffness of the glass slide was determined (figure 3E). We found that cells covered with a layer of polysilicate have a stiffness of 0.43 compared to the glass slide and the cells without a layer of polysilicate have a stiffness of 0.16 compared to the glass slide. We can see that due to the encapsulation of the cells in polysilicate the stiffness of the cells increases significantly.


Concluding, we have characterized this part using different techniques and we have demonstrated that it works as expected: it covers the E. coli in a polysilicate layer. For more information about these experiments visit our project page.

References

  1. 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
  2. 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.
  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.
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