Team:TU Delft/Project
Opticoli
Synthetic biology to enable biological lenses and lasers
Project Description
BIOLASERS
Imaging cells is essential for understanding life at the smallest scale and fighting cellular diseases like cancer. Imaging often relies on fluorescence, but fluorescent proteins have some drawbacks, such as their wide spectrum and low intensity.
Our biolasers will provide an accurate, safe and biological way to improve this.
Fluorescence is the ability of a molecule to take up the energy of a photon and release it again, which makes the molecule light up. Lasing works with the same principle as fluorescence, but now the light source is put between mirrors. The photons keep ‘bouncing’, increasing the energy of the system. When the light gets a certain power, the photons can escape in the form of a laser beam.
A biolaser is achieved by trapping fluorescent proteins inside a reflective agent. We have chosen two reflective agents: bioglass (polysilicate) and bioplastic (PHB). By covering a cell with polysilicate, the photons can resonate inside the cell, making a whole-cell laser. The polysilicate is synthesized by an enzyme called silicatein, which is expressed on the cell wall by fusion to membrane proteins. By filling a cell with PHB, which forms intracellular granules, the photons can resonate inside a part of the cell, making an intracellular laser. The PHB is synthesized after expressing the pha-operon. By fusing the GFP to the PHB synthase, the GFP is relocated into the PHB granules.
BIOLENSES
Microlenses are an emerging field in technology and have a ton of applications, including high-tech cameras, chips, solar panels and research & imaging techniques. However, they are expensive, hard to fabricate and the production uses heavy chemicals and high temperatures, so it is bad for the environment.
Our biological microlens will be cheap, easy to make and environmentally friendly.
When we cover a cell with polysilicate, using the enzyme silicatein, we are able to make a biological microlens. By overexpressing either the transcriptional regulator bolA or the cell division inhibitor sulA we can play with cell morphology and investigate optical properties. These enlarged cells can also be used in the lasing experiments. The single cell will be able to diffract light as a single microlens. When we make a grid of lenses, a microlens array, we can use the lens for a coating for solar panels, thin lightweight cameras with high resolution or 3D screens.
Experiments and results
Expression of different fluorophores
Introduction
One of the essential components of a laser is a fluorescent agent. Since our aim is to produce a fully biological laser, fluorescent proteins are favourable. To this end, we initially selected four fluorophores, with different emission wavelengths: GFP, mVenus, mKate, mCerulean. These fluorophores were reported to have an increased fluorescent intensity compared to their wildtype.(SOURCE). In order to characterize them before further use, the emission and absorption spectra were measured. Additionally, their effect on cell growth was investigated.
Experiments & Results
Before continuing to work with the fluorophores, their fluorescence was validated by measuring the emission and absorption spectra. The fluorophores were expressed in E. coli BL21 and they were characterized in a plate reader.
Emission spectra of different fluorophores
Introduction
The emission spectrum of each fluorophore was determined by exciting at a given wavelength and measuring the output intensity at a range of wavelengths. Because of this, the emission at a wavelength too close to the excitation could not be measured.
Methods
Parts encoding the four different fluorophores, including promoter, RBS and terminators, were expressed in E. coli BL21. GFP, mVenus, mKate and mCerulean were all expressed under the same strong constitutive promoter, J23100. Additionally, parts were constructed with GFP under control of promoters with different strengths, in order to investigate the influence of different fluorophore concentrations.
After growing in LB medium the cells were washed and resuspended in PBS of which aliquots of 100 µL were put in a 96 well plate.
Results and Discussion
As all fluorophores were expressed under a strong constitutive promoter, they were expected to show a strong fluorescence without the need of induction. Figure X shows that this was the case for GFP, mVenus and mCerulean. mKate, however, did not show any fluorescent activity and was therefore not used in the subsequent steps of the project.
Back to TopExpression & Characterization of our GFP collection
Place experiment (Introduction, methods, results&Discussion here)
Back to TopDiscussion & Conclusions
Conclusion and discussion on the experiments
Coating the cell in polysilica using silicatein
Introduction
Introduction on silicatein & experiments
Experiments & Results
Introduction on the experiments that we did
Rhodamine staining of silica covered cells
Place experiment (Introduction, methods, results&Discussion here)
Back to TopImaging of silicatein-expressing cells using SEM
Place experiment (Introduction, methods, results&Discussion here)
Back to TopImaging of silicatein-expressing cells using TEM
Place experiment (Introduction, methods, results&Discussion here)
Back to TopAnalysis of physical properties of silica covered cells using AFM
Place experiment (Introduction, methods, results&Discussion here)
Back to TopViability experiments of silica covered cells
Place experiment (Introduction, methods, results&Discussion here)
Back to TopDiscussion & Conclusions
Conclusion and discussion on the experiments
Engineering a biological laser
Introduction
Our cellular laser consists out of two features: fluorophores will be the light of the laser and a silica layer synthesized by silicatein will be the ‘mirrors’ that reflect a part of the photons emitted by these fluorophores. The fluorophores first need to be excited by an external light source. This could be either an LED source or a conventional solid laser. For fluorescence an LED excitation source suffices. However, we want lasing to happen in our cells. For this to happen, we need ‘population inversion’, which means the majority of the fluorophores is in an excited state (Gather & Yun, 2011; Svelto & Hanna, 1976). More information on this can be found in the project description. In order to excite the majority of the fluorescent proteins at the same time, we need a strong excitation source. Therefore we need to use a laser to excite the fluorphores in our biolaser. However, a major downside to using lasers for fluorophore excitation is the occurrence of photobleaching (Eggeling, Widengren, Rigler, & Seidel, 1998). The laser power required for the excitation of fluorophores to induce population inversion in the cell is so high it will photobleach the fluorophores within microseconds (Jonáš et al., 2014). Therefore, we need a custom laser setup to prevent photobleaching but establish the population inversion required for our biolaser. This laser setup should contain a pulsing laser which pulses at a frequency that will maintain the excited state but does not photobleach the proteins.
The appropriate set-up was not available, so we decided to build our own microscope out of separate optical parts. By discussing our problem with optics- and photonics companies and showing our motivation to solve this challenge, we were able to get all our required components sponsored or borrowed, making the entire set-up nearly cost-free. With our minimal set of available tools, we calculated and designed the optics in such a way that we could image fluorescent cells, while photobleaching was minimized. After days of laser aligning, we managed to do so. More information on the design of this setup can be found on the hardware page.
Using our custom-built setup, we analysed our ‘Biolaser’-cells, to see if they were able to produce a laser-like emission of light.
Experiments & Results
The first and foremost experiment to be done with the setup was to image the cells in order to see if the setup works properly and to see whether we can image cells with it. Once we have confirmed that the setup works properly, we can measure the output intensity of the cells to see whether the cells are able to emit laser-like light.
Imaging our biolaser using our homebuilt setup
Introduction
Building an optical setup is a very precise work. First, it is important to calculate the positions of all components in such a way that the laser beam will reach your sample, and the light emitted by the sample consequently reaches the detectors of the camera and spectrometer. Once this is done, all optical components are positioned on an optical table. This special table is made to prevent vibrations in you system and has mounting holes so all optical components can be screwed into place. Once the components are positioned on the table, the alignment begins. In this step, the beam coming from the laser is guided throughout the system. By slightly adjusting and repositioning all optical components the light is guided through the components until it reaches the detector of the camera. It is essential that the components are aligned correctly and are free of vibrations, because this could change the path of the light.
After tens of hours of carefully placing components and aligning the light through the setup, we managed to direct the light from the laser, through all components onto the detector of the camera. However, this does not necessarily mean that when we add fluorescent cells to the setup it we are able to image the cells with the setup. Any error in the setup could cause it not to work. Therefore, we first had to confirm whether our setup was working.
Methods
In order to confirm whether the setup was working, we used E. coli BL21 cells that were transformed with our constitutive mCerulean BioBrick. We have previously confirmed that these cells are able to fluoresce and that they can be excited at 405nm, the wavelength of our laser. To make sure the only output we were measuring was fluorescence, and not any ‘leakage’ of light from our laser beam, we also tested cells that were not transformed with the mCerulean BioBrick. These cells are not able to fluoresce after excitation at 405nm, so if the setup is working properly we should not get a signal from these cells.
The cells were fixated to the microscope slides using 3% agarose pads and imaged at an excitation intensity of 0.5 mW. This energy is low enough to not instantly photobleach the proteins, but observe fluorescence clearly. Focusing on the cells was done manually with a 50x oil-immersed objective. The cells were excited with a Coherent OBIS LX 405nm laser and images were taken using a DeltaPix Invenio III CCD camera.
Results & discussion
Imaging the cells with the setup yielded the following results:
As we can see in figure 2B, the cells transformed with a gene for the fluorescent protein mCerulean are clearly visible. When using a strain transformed with a plasmid that is not known to cause the cells to fluoresce, in this case OmpA-silicatein, no light was observed, as shown in figure 2A. From this we can conclude that we successfully built a setup that is able to observe and measure fluorescence in a cell. There is no leakage of light of our excitation laser in the camera, since we do not observe anything when we use non-fluorescent cells. Also at a higher excitation energy (50 mW) we did not observe anything on the camera. Therefore we can conclude that our setup works as expected as it is indeed able to measure fluorescence without measuring other light sources.
For this experiment we used E. coli transformed with OmpA-silicatein that was induced and incubated in silicic acid, so it would contain the silica layer. We used this strain both as a negative control as well as to test whether the silica or the cells had any autofluorescence that could interfere with our laser experiments. We did not observe any fluorescent signal for these cells, so we can conclude that the silica layer does not have any autofluorescence at 405 nm. Therefore, these cells are suitable for the laser experiments.
Back to TopIntensity measurements of laser cells
Place experiment (Introduction, methods, results&Discussion here)
Back to TopDiscussion & Conclusions
Conclusion and discussion on the experiments
Engineering biological lenses
Introduction
Introduction on lenses & experiments
Experiments & Results
Introduction on the experiments that we did
Influencing cell shape for round lenses
Introduction & background
When making biological lenses, the shape of the lens is of crucial importance. E. coli is a rod-shaped organism, so it’s 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, see figure 1. More information on this can be found on the modeling page.
For some applications, such as the solar cells, this variation in shape does not matter that much; here it’s most important that light gets focused in any way. However, when we want to use our microlenses in more advanced optical systems, such as microscopes or cameras, we need to make sure that this variation between the different lenses is minimized. Manufacturers of optical systems do not accept a high aberration between different lenses, so it’s crucial for us to be able to control the shape of our lenses. We have decided to engineer E. coli in such a way that it becomes spherical. This way we are able to create spherical lenses. Apart from the fact that it is crucial to be able to control cell shape, round cells offer the advantage of being symmetrical along all axes, so the orientation of your lens does not matter for the optical properties.
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, Freire, Vicente, & Arraiano, 1999). By overexpressing this gene, the rod-shaped E. coli cells will become round (Aldea, Hernandez-Chico, De La Campa, Kushner, & Vicente, 1988). When we express both the BolA gene as well as silicatein, we are able to construct round cells, coated in glass.
Methods
The phenotype of the cells expressing BolA is very different from the phenotype of wildtype E. coli. If the gene is successfully overexpressed, the cells become round, which we can easily observe under a widefield microscope. In widefield microscopy, the whole sample is simultaneously illuminated using a white light source so the phenotype of the sample can be inspected. This is comparable to normal light microscopy.
In order to obtain round cells we tested transforming E. coli BL21 with BolA under both an inducible promoter (Lac) and a constitutive promoter (J23100). Furthermore, we tried if transforming a strain with both BolA and the OmpA-silicatein fusion plasmid yielded round, glass covered cells. The following strains and conditions were tested under the widefield microscope:
| Plasmid(s) | IPTG | Silicic acid |
|---|---|---|
| Lac-BolA (inducible) | - | - |
| Lac-BolA (inducible) | + | - |
| J23100-BolA (constitutive) | - | - |
| OmpA-Silicatein (T. aurantia) + Lac-BolA (inducible) | + | + |
The cells were heat-fixed on a slide and observed under the widefield microscope.
Results and discussion
The four different strains were imaged under the widefield microscope, the taken images are shown in figure 2.
In figure 2, the widefield images of the four tested strains are shown. We can see from figure 2A that solely transforming E. coli with BolA but not inducing the plasmid results in cells with the phenotype of wildtype E. coli; the cells are rod-shaped. Figure 2B shows that induction of the cells transformed wil BolA under the inducible Lac-promoter indeed has changed the phenotype of the cell. The cells have clearly become spherical. Constitutive expression of the BolA gene, as shown in 1C has the opposite result: the cells are rod-shaped and elongated. This is probably because of the stress the plasmid puts on the cells. As mentioned before, BolA is a gene involved in the stress response of E. coli that changes the morphology of the cells. A too high expression of the gene could therefore change the morphology in an unexpected way, such as elongation, a phenomena that is often observed in E. coli (Höltje, 1998). Since we require sphere-shaped cells, constitutive expression of the BolA-gene is not desired. Co-expression of the OmpA-silicatein fusion plasmid and the inducible BolA plasmid also yielded round cells, as seen in figure 2D.
So, by inducing the expression of BolA, we are indeed able to control the shape of E. coli and turn the cell into a sphere. Also, transforming a cell with both silicatein-OmpA and BolA yields round cells, so the formation of the glass layer does not distort the cell shape. The glass layer can not be seen under the widefield microscope, but we previously confirmed the presence of the silica layer with AFM and rhodamine staining. Being able to control cell shape is of major importance if we want to create biological lenses, since the lenses are desired in various sizes and shapes. Especially spherical lenses are useful since they are symmetrical and therefore do not require a specific orientation; they will focus the light in the same way whatever their orientation is. From figure 2 we can see that the ells are not perfectly homogeneously shaped, there is some variation between the shape of the different cells. This variation is even clearer for the cells that contain the silica layer. This is possibly because two plasmids with a lac promoter put a great strain on the cells, resulting in a greater variation. For precision optics, it is extremely important that there is little to none variation between the lenses. Therefore, it’s recommended to do more research in controlling cell shape. However, since there is always a variation in gene expression between cells it will be wiser to conduct research into selecting or sorting the cells on their size or shape. A possible solution could be sorting the cells using FACS. As a future experiment, we could stain the silica-coated cells with Rhodamine and let a FACS-machine sort the cells on their phenotype.
Back to TopRemoving the cells from the biolenses
Place experiment (Introduction, methods, results&Discussion here)
Back to TopImproving solar cells with biolenses
Place experiment (Introduction, methods, results&Discussion here)
Back to TopDiscussion & Conclusions
Conclusion and discussion on the experiments
Conclusions & Recommendations
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.
- Eggeling, C., Widengren, J., Rigler, R., & Seidel, C. (1998). Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis. Analytical Chemistry, 70(13), 2651-2659.
- Gather, M. C., & Yun, S. H. (2011). Single-cell biological lasers. Nature Photonics, 5(7), 406-410.
- Höltje, J.-V. (1998). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiology and Molecular Biology Reviews, 62(1), 181-203.
- Jonáš, A., Aas, M., Karadag, Y., Manioğlu, S., Anand, S., McGloin, D., Kiraz, A. (2014). In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities. Lab on a Chip, 14(16), 3093-3100.
- 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.
- Svelto, O., & Hanna, D. C. (1976). Principles of lasers: Springer.
