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iGEM TU Delft

The new age of optics: Producing biological lenses and lasers to improve microscopy

Project Description

Limitations of microscopy

Microscopes have been around for hundreds of years and the technology behind these devices has been quickly developing over the past centuries. Especially fluorescence microscopy was an essential discovery for us biologists, since we are especially interested in what processes occur inside the cell. A popular approach to image intra- and extracellular processes is to use fluorescent tags to track a molecule- or gene of interest in the cell. These fluorescent tags can be imaged under a fluorescence microscope, allowing us to trace molecules and gene expression in a cell.

This popular technique has been essential for cellular research over the past decade, and has helped us to find out several important basics of life. Even though most techniques are already very far developed, it is essential that we keep developing microscopy techniques even further. When we can image every process going on in the cell, we are able to use this for our own good. We can, for example, image the mode of action of a disease, and cure this. An example of a disease that has been studied for years but still not fully understood, is Alzheimer’s disease (Hardy & Selkoe, 2002). If we could tag all molecules in a brain cell and image them, we might find what exactly causes the disease and hopefully develop a treatment. Also synthetic biology in general benefits from a good understanding of the cell. When we can trace all enzymes involved in a certain process, for example the alcoholic fermentation in yeast, we can more easily modify genes of interest and optimize this pathway for an improved production of biofuels.

Unfortunately, microscopy hasn’t advanced that far yet. Though super-resolution microscopy is quickly developing, there are still several limitations that hinder a full visualization of the cell. At this point, the technology and knowledge of microscopy is not the biggest limit for making detailed images of the cell; it’s the cells itself. When using fluorescence microscopy, the limit of the resolution of the microscopy is the amount of photons that your sample emits. However, not all photons are observed by the detector of the microscope, simply because not all photons reach this detector and get lost in the noise of the detector (Heintzmann & Ficz, 2006). Especially in tracing low intracellular concentrations or high-speed cellular processes, the amount of photons emitted is low (Lakowicz, 2013). We aim to improve this limit of microscopy using synthetic biology.

**Figure 1,** When a fluorescent cell is imaged, not all photons will reach the detector, so not all photons will be detected.

Improving microscopy using biology

The chance of a photon being observed is defined by both the chance of the photon being emitted and the photon being detected (Heintzmann & Ficz, 2006). We have thought of two possible solutions to improve the observation of a photon. The first one is increasing the chance of a photon being emitted by the sample, which in this case, are our cells. We will do this by modifying the cells in such a way they are emitting more photons for the same amount of fluorophores. This technique is based on the working principle of a laser. Therefore, we will call these cells the Biolaser.

A second approach to increase the chance of a photon hitting the detector is by directing the photons towards the detector. An easy way to do this is to apply a layer of lenses over the detector to focus the light onto the detector. A layer of tiny lenses is also called a microlens array. However, these microlens arrays are hard to fabricate and their synthesis is harmful for the environment. Therefore, we will modify bacteria in such a way they will become lenses: the Biolenses.

**Figure 2,** two ways to improve the amount of photons detected in fluorescence microscopy. First, by modifying a cell so it emits more photons using the same amount of fluorophores, we increase the chance of detecting a photon. This method is based on the working principle of a laser and is therefore called a Biolaser. Secondly, using an array of lenses we can focus light onto the detector, increasing the amount of photons detected. These lenses are synthesized using microorganisms, hence they are called Biolenses. Click on the images to find out more.

**Figure 2,** two ways to improve the amount of photons detected in fluorescence microscopy. First, by modifying a cell so it emits more photons using the same amount of fluorophores, we increase the chance of detecting a photon. This method is based on the working principle of a laser and is therefore called a Biolaser. Secondly, using an array of lenses we can focus light onto the detector, increasing the amount of photons detected. These lenses are synthesized using microorganisms, hence they are called Biolenses. Click on the images to find out more.

Synthetic biology to produce biological lasers

In order to modify E. coli in such a way that it is able to emit laser-like light, we first need to understand how a laser works. A laser has three essential components: a gain medium, an excitation source and a reflective agent. The gain medium is a medium with the ability to fluoresce. This gain medium is surrounded by a reflective agent, such as a mirror. This gain medium gets excited by the excitation source, which could be either an electric pulse or an external light source. Once this gain medium gets excited, it will emit photons. Because the gain medium is surrounded by a mirror, the photons cannot escape the gain medium but will ‘bounce’ back. When one of these photons hits an excited fluorescent molecule, something remarkable happens. This molecule will release an exact copy of the incident photon. This process is called stimulated emission. The result is that the light gets amplified each time it passes through the gain medium. Therefore, with only a limited amount of fluorescent molecules we can emit a lot more light compared to ‘conventional’ fluorescence.

In this project, we will use synthetic biology to modify E. coli in such a way that the bacterium will be able to emit laser-like light. In order to do this, we have to translate two of the main components of a laser, the gain medium and the mirrors, to biological alternatives that E. coli is able to produce. For the gain medium this is easy. We can transform E. coli with fluorescent proteins that will form the fluorescent gain medium. The biological alternative for mirrors is slightly less obvious. However, we believe to have found the solution in the enzyme silicatein. This enzyme is able to synthesize polysilicate, a biological glass (Müller et al., 2008; Müller et al. 2003). By transforming E. coli with the gene for this enzyme, we can let the cells coat itself in a layer of glass that will reflect the photons emitted by the fluorescent proteins. As an excitation source we can simply use a laser to excite the proteins. A figure of our synthetic biology approach to create a biological laser can be seen in figure 4. This biolaser will be able to emit a higher number of photons compared to a cell that is merely fluorescent. Therefore, this cell will be useful in fluorescence microscopy, since even a cell with a low number of fluorophores is hypothetically able to emit a high number of photons. Therefore, this cell could be clearly imaged. Our parts, experiments and results can be found in the Experiments section

**Figure 4,** designing a laser using synthetic biology. The lasers gain medium can be substituted by fluorescent proteins, a layer of biologically synthesized polysilica acts as the mirror around the gain medium. The fluorophores are excited by a laser.

Synthetic biology to produce biological lenses

The second approach to capture more light in fluorescence microscopy is by focusing the photons onto the detector of the microscope. We will do this with lenses, since they have the ability to focus light onto an object, such as the detector of a microscope, which is schematically shown in figure 5.

**Figure 5,** When shining a beam of light onto a detector, not all light might hit the detector. Placing a lens in front of the detector focuses the beam onto the detector, allowing the detector to measure all photons.

In a microscope, applying this technique would mean that each photovoltaic cell of the detector gets a lens placed on it that will direct all light into each cell of the detector. This means we need a matrix, or “array”, of lenses that are only a few micrometres small. These ‘microlens arrays’ already exist, and have been shown to be a good technique to focus more light onto photovoltaic cells, including the detector of a microscope (Jutteau, Paire, Proise, Lombez, & Guillemoles, 2015). We will also use this technique. However, we will not use the conventional, chemically produced microlenses, since they are very costly and their production is difficult and bad for the environment (Nam et al., 2013). Therefore, we aim to produce our own, biological microlenses that will be much greener and environmentally friendly compared to the conventional microlenses. Furthermore, we will also apply our biological microlenses in applications, other than microscopy, where collecting light is important. One of these applications in which we will apply our biological lenses are solar cells, more information can be found on the practices page

So how do we aim to produce these biological lenses using synthetic biology? The approach is the same as for the biological lasers: we transform E. coli with the silicatein gene. This gene allows the cell to cover itself in glass, resulting in a glass sphere of only 1-2 µm small. We will test the optical properties of this glass sphere as well as its functionality as a microlens. Since the shape of lenses is of high importance, we will also research ways to manipulate the size and shape of the cells, enabling us to produce different shapes of microlenses. Also, since our biological microlens contains a core of live bacteria, we will look into ways to sterilize the lens. This way, our product will not harm the environment in any way. Our parts, experiments and results can be found in the Experiments section

**Figure 6,** By transforming *E. coli* with a gene for the enzyme silicatein, the bacterium is able to coat itself with polysilicate, a kind of biological glass. This turns the bacterium in a glass sphere of 1-2 µm that can function as a microlens.

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 and mCerulean. These fluorophores were reported to have an increased fluorescent intensity compared to their wildtype (Cormack et al., 1996; Nagai et al., 2002; Shcherboo et al., 2007; Rizzo et al., 2004).

Since mVenus, mKate and mCerulean did not exist in the iGEM registry yet, we constructed a brand new part for each of these fluorophores including strong constitutive promoter and RBS (parts K1890010 and K1890011). GFP, on the other hand, was present in a whole range of biobricks. However, to our knowledge, there was no single biobrick available containing promoter, RBS and terminators. Hence, we constructed a new biobrick containing all of the above using the existing part E0840, consisting of RBS, coding sequence and terminators. By means of PCR we amplified this biobrick with primers designed to add a promoter while mainaining the biobrick prefix and suffix. Not only did we express GFP under the strong promoter J23100, but also under less strong promoters J23113, J23117, J23105, and J23108. This way we were able to see the influence of promoter strength on fluorescent output (parts K1890020 to K1890024.

Experiments & Results

In order to characterize these biobricks before further use, the emission spectra of the fluorophores were measured. Additionally, their effect on cell growth was investigated.

Emission spectra of different fluorophores

Introduction

To assure the fluorophores were functional, the emission spectra were recorded at the given excitation wavelength.

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.

All of the mentioned biobricks were expressed in E. coli BL21. After growing in LB medium to an OD600 of ~1, the cells were washed and resuspended in the same volume of PBS. Aliquots of 100 µL were put in a 96 well plate.

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. This can be seen in the figures, where the left half of the emission peaks could not be measured. Especially for mVenus, where the excitation and emission wavelengths are very close together.

Results and Discussion

Fluorophores expressed under strong constitutive promoter

As all fluorophores were expressed under the strong constitutive promoter J23100, they were expected to show a strong fluorescence without the need of induction. Figure 1 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.

GFP expressed under constitutive promoters of different strengths.

Not only did we express GFP under the strong promoter J23100, but also under less strong promoters J23113, J23117, J23105, and J23108.

For comparison, the emission was normalized by dividing by OD. All strains were measured in the same dilution, in order to make the results reproducible. The emission intensity is as expected, corresponding to the strength of the promoters. All fluorophore spectra were also recorded in a dilution more suited for their emission intensity and normalized by 1 (Figure 3). From this figure we can conclude that all GFP biobricks function properly.

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The effect of expression of fluorophores on growth

Introduction

As consitutive expression can sometimes be hard on the cells, we investigated the effect of the different consitutive promoters on cell growth. In a 24 hour kinetic cycle alterating shaking at 37°C with fluorescence and optical density measurements, we investigated whether this was the case.

Methods

An overnight culture in eM9 medium was inoculated in fresh eM9 to an OD600 of 0.1 in a 96 well plate. The emission at 522 nm was measured every 15 minutes. Measurements were done in quadruplicate with pure eM9 as a blank.

Results and Discussion

Figure 1 shows the 24 hour measurement of optical density and fluorescence intensity. The final OD600 is approximately equal for all different strains, suggesting that the level of constitutive expression was not influencing the growth. Furthermore, fluorescence intensity drops after the exponential growth phase, suggesting that GFP is being broken down by proteases as a response to nutrient limitation. After this event, growth continues at a slower pace, while GFP activity keeps decreasing. All in all, constitutive expression of GFP does not seem to have a detrimental effect on cell growth during exponential phase.

Cells expressing mCerulean or mVenus, however, seem to be having a longer lag phase before exponential growth starts (Figure 2). Also, they reach a lower final OD600 than the ones expressing GFP. These fluorophores might be slightly harmfull for cell growth. Nonetheless, they do grow and exhibit fluorescence, so they can be used in further experiments.

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Discussion & Conclusions

We were able to succesfully construct and characterize two biobricks with brand new fluorophores for the iGEM registry: mVenus and mCerulean. Both of them exhibit a bright fluorescence, which is useful in our futher experiments. To our knowledge there was no part available in the registry consisting of promoter, RBS, GFP coding sequence and terminator Therefore, we constructed five new composite parts to provide single complete biobricks for GFP expression. These new parts were characterized and show a bright fluorescence, according to their promoter strength. All in all, our new parts provide a ready-to-go expression device for green, yellow, or cyan fluorescence, which can be very usefull for future iGEM teams.

Coating the cell in polysilica using silicatein

Introduction

For both the biolaser and the biolenses we need a coating of polysilicate, biological glass, around the cell. For the biolaser this glass will form the cavity that will enable the cells to emit laser-like light. For the biolens, the 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 the use of two different silicateins, one originating from the marine sponge Suberites domuncula (Müller, 2011) and one originating from the marine sponge Tethya aurantia (Cha et al., 1999). We express the enzyme in three different ways. First of all, we expressed the gene from S. domuncula (Part K1890000) and see if the enzyme is transported outside the cell as described by Müller et al, 2008. Furthermore, we express a fusion of silicatein from T. aurantia to the trans-membrane protein OmpA (outer membrane protein A) from E. coli to anchor the silicatein to the membrane (Part K1890002) (Curnow, Kisailus, & Morse, 2006; Francisco et al. 1992), which might make coating the cell specifically in polysilicate more efficient. We also express a fusion of silicatein from S. domuncula to the transmembrane Ice Nucleation Protein (INP) from Pseudomonas syringae (Part K1890001), a popular protein for membrane fusions (Kim & Yoo, 1998). Using these different approaches we expect to coat the cell in polysilicate, an overview is shown in figure 1. This glass coating around the cell will be the basis of both our biolens and –laser.

**Figure 1,** (A) Silicatein is able to convert monosilicate to polysilicate, allowing the cell to cover itself in polysilicate. (B) We express silicatein in three ways: solely expressing silicatein and fusing it to the membrane proteins OmpA or INP.

Experiments & Results

To determine if we have successfully covered E. coli in polysilicate, and to characterize the properties of the silicate-coated cells, we have performed a series of tests. First of all, we have stained the cells with rhodamine, a fluorescent stain that is able to bind to polysilicate. These stained cells were observed under a fluorescence microscope to determine whether the polysilicate shell was present. Furthermore, the polysilicate-synthesizing cells were observed using both Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). We have also determined the physical properties using Atomic Force Microscopy (AFM) to see whether the polysilicate layer changes the stiffness of the cells. Lastly, we have also performed a growth study of the polysilicate-coated cells to determine whether the polysilicate layer affects growth of the organisms.

Rhodamine staining of silica covered cells

Introduction

After transforming E. coli with the different silicatein BioBricks, we wanted to confirm and image whether the cell had indeed synthesized a layer of polysilicate around itself. Before using any advanced imaging techniques, we first used a much simpler technique. It is possible to stain polysilicate depositions with the fluorescent stain Rhodamine 123. This stain has shown to bind specifically to polysilicate (Li, Chu, & Lee, 1989). Since the stain is fluorescent, we can image it with a simple fluorescence microscope. If a cell has a polysilicate layer, the Rhodamine will bind to it which we can image.

Methods

The experiment was performed using E. coli BL21 with the following plasmids and conditions. All genes are under an inducible promoter.

Plasmid(s)	IPTG	Silicic acid	Rhodamine 123
OmpA-Silicatein	+	+	+
Silicatein	+	+	+
INP-Silicatein	+	+	+
OmpA-Silicatein (negative control)	+	-	+
OmpA-Silicatein	+	+	-

The cells were stained with 0.1 vol% Rhodamine. And washed 5 times with PBS, prior to imaging (Li et al., 1989; Müller et al., 2005). Both widefield- (light microscopy) and fluorescence microscopy were used to image the cells.

Results and discussion

The stained cells were first imaged at maximum excitation intensity. At this excitation energy, only OmpA-silicatein showed fluorescence specifically localized at the cells and not in the medium. Silicatein, INP-silicatein and the negative control all caused overexposure of the camera. In figure 1 the imaging results of OmpA and the negative control are shown. The cells that were not stained with Rhodamine showed no measurable fluorescence. (data not shown).

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.

Since Silicatein, INP-silicatein and the negative control all caused overexposure of the camera, they all had the same output. We can thus not draw any conclusions for these strains. Therefore, samples where overexposure was observed were imaged again at only 1/3 of the excitation energy. The imaging results are displayed in figure 2.

At this excitation energy, we can compare these samples. From figures 1 and 2 we can see that the strain transformed with OmpA-silicatein clearly has a different output from the negative control. The fluorescence is only localized at the cells. From this we can conclude the Rhodamine has stained the cells and therefore these cells will contain the polysilicate layer. We cannot distinguish a clear difference between silicatein, INP-silicatein and the negative control. The entire medium is fluorescent, which causes overexposure of the camera at high excitation intensity. This might mean that the Rhodamine is not specifically located at the cell walls, but still dissolved in the medium. Wedo see some fluorescence localized at the cells, but the diference between the fluorescence of the medium and the cells is much smaller than we observed for OmpA-Silicatein. Therefore, we cannot conclude that the strains transformed with silicatein and INP-silicatein are able to synthesize a polysilicate layer around the cell. We might be able to test this using SEM or TEM, but from this test we can not draw a conclusion for these two strains.

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Imaging of silicatein-expressing cells using TEM

Introduction

Electron microscopy is an imaging technique that can acquire images with resolutions far above the optical limit due to the small wavelength of electrons (Nellist, 2011). An electron gun forms an electron beam which is focused and accelerated by magnetic lenses. When the electrons reach the sample, there are several interactions possible with the sample (figure 1). These various interactions can be used for different techniques. We have used high-angle annular dark-filed transmission electron microscopy (HAAFD-TEM) to make an image of the sample, energy dispersive x-ray spectroscopy to analyze the element composition of the sample and scanning electron microscopy (SEM) to analyze the phenotype of the samples.

**Figure 1:** Interactions between the electron beam and sample in electron microscopy (slideshare, 2013).

In HAAFD-TEM electrons pass through the sample and are inelastically scattered at a high angle and acquired by a detector. The amount of electrons scattered at a high angle depends on the thickness and the material of the sample. When the thickness is high, the sample scatters a lot it will appear bright in the image.

Energy dispersive x-ray spectroscopy (EDX) is an analytical technique to determine the elements present in the sample (Friel et al. ,2006). When the electron beam interacts with the sample x-rays can emerge from the sample. These electrons can be detected and are characteristic for the element which emitted the x-ray. In that way we can specifically determine which elements are present in our sample.

Methods

The experiment were performed using E. coli BL21 cells with the plasmid containing OmpA-Silicatein. Two samples were made where the first sample was induced with IPTG but no silicic acid was added and a second sample which was both induced with IPTG and silicic acid was added, therefore this sample will have a polysilicate layer. The samples were prepared by fixation using 1% polylysine on a quantifoil carbon grid.

Results and discussion

Both samples were imaged using HAAFD-TEM and energy dispersive x-ray spectroscopy (figure 2). The cells are fixed at a carbon grid. In each sample, we measured the silicon content. We can clearly see that the grid contains silicon and there is a lot of silicon signal from the grid (figure 2 B,D), since each blue dot represents silicon being measured. When a cell is positioned at a hole on in the grid we there is no background silicon signal, so we can observe the elemental composition of our sample. For the sample where no silicic acid is added, we can see some silicon present at the cell position. However, there is a significant increase in silicon detected for the sample where silicic acid was added to the sample. This shows that silicon co-localizes with the cell which means there is indeed a polysilica layer formed by the bacteria.

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Imaging of silicatein-expressing cells using SEM

Introduction

The Scanning Electron Microscope, or SEM as it is called, it is one of the most widely used imaging techniques in science. It is highly used due to its very high spatial resolution and Depth of Focus (DoF). SEM can reach resolution of 5 nm and DoF about 0.5 mm, compared to an optical microscope that can reach a spatial resolution of \( 0.2 \mu m \) and DoF \( 1 \mu m \). Important here is to note that with SEM it is only possible to image solid samples because the measurements take place under vacuum. Since we want to image the impact of the polysilicate layer in the highest detail, we use sem to image our samples.

In SEM a beam of electrons is accelerated by high voltage and it is directed towards the sample with speeds up to \( 1.64 \times 10^5 km\). When the beam hits the sample, x-rays, backscattered electrons and secondary electrons are generated. For imaging, SEM uses the secondary electrons. Those secondary electrons are then collected and their kinetic energy is transformed to an image. (Kwakernaak & Sloof, 2005)

Methods

The polysilicate layer around the cells was prepared according to the polysilicate layer protocol. As a negative control, uninduces cultures (no IPTG) and cultures without substrate (no sodium silicate) were used. The samples were fixed with gluteraldehyde and imaged with SEM.

Results and Discussion

Figure 1 shows SEM images taken of samples in the presence (A, B) or absence (C, D) of silic acid. First of all, since we know from the rhodamine staining experiment that the polysilicate layer is present, it does not seem to influence the cell shape. However, the cells that are expected to have a polysilicate layer appear to be somewhat fused together. According to Müller et al (2008), cells possessing a polysilicate layer appear to be fused by a viscous cover. This can, however, also be the result of limited imaging resolution. When using titanium oxide as a substrate, Curnow et al (2005) reported large aggregates visible by SEM. This was not observed in the current experiment. Therefore, we can conclude that the polysilicate layer does not form aggregated or influence the shape of the cell, but form a homogeneous layer around the cell.

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Analysis of physical properties of silica covered cells using AFM

Introduction

Due to encapsulation of the cell with a layer of polysilica the stiffness could change. We wanted to know whether and how the stiffness changes once e call is coated in polysilicate and determined this using Atomic Force Microscopy (AFM).

AFM is a technique used to determine surface characteristics of a sample. In AFM a very sharp tip scans over the surface of the sample. While scanning over the surface every height change is detected by a laser which is reflected by the cantilever on a position-sensitive photodetector (Figure 1). In the PeakForce QNM mode, ehich we used, also surface characteristics like the stiffness and deformation can be determined. The stiffness is computed from the relation between the force at which the cantilever pushes on the sample, the adhesion force and the deformation of the material.

**Figure 1:** AFM Setup. A laser is reflected by a cantilever and detected by a position sensitive photodetector. Every change in height of the sample results in a change of height of the cantilever and is measured.

Methods

Experiments were performed using E. coli BL21 cells transformed with the plasmid containing the OmpA-Silicatein gene. Silicic acid was added to one sample to form the polysilicate layer around the cell. Another sample with no silicic acid added was used as a control. The cells weres spun down and resuspended in MilliQ and fixated on a glass slide using 1% ABTES.

Results and discussion

We have imaged two samples with AFM. The first sample is OmpA fused with silicatein with silic acid added so that the cell can encapsulate itself with biosilica (figure 2A-B). The second sample, that wa used as a control, did not contain silicic acid (figure 2C-D). From both samples a height map (figure 2A,C) and the stiffness (figure 2B,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 stiffness of the glass slide of both samples.

Multiple cells were imaged and the relative stiffness of the cell compared to the stiffness of the glass slide was determined (figure 3). 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 polysilica the stiffness of the cells increases significantly.

**Figure 3:** 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.

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Viability experiments of silica covered cells

Introduction

Since the silicatein expressing cells are to cover themselves in polysilicate, their nutrient supply might be limited by diffusion, which can eventually result in cell death. To investigate whether this is indeed the case,a growth study was performed.

Methods

Cells containing the three different silicatein biobricks were grown overnight in selective LB. They were transfered to fresh medium and grown until in exponential phase. Then IPTG was added to induce expression. After a subsequent incubation of three hours, the medium was supplemented with silicic acid as substrate for silicatein. During the following five hours samples were taken, of which a 10^-6 dilution was plated on selective LB plates. Colony forming units (cfu) were counted the day after.

Results and Discussion

Cells expressing either silicatein from S. domuncula (Sil Sdom), silicatein from S. domuncula fused to INP (INP Sil Sdom) or silicatein from T. aurantia fused to OmpA (OmpA Sil Taur) were tested. As a negative control, OmpA Sil Taur expressing cells without silicic acid were used. After one hour no colonies were observed on the plates on which the cultures with silicic acid were plated (Figure 1). The cultures without silicic acid continued to grow until after five hours.

Hardware setup — **Figure 1,** Number of colony forming units (cfu) during incubation with silicic acid.

This figure suggests that either the polysilicate layer inhibits nutrient diffusion into the cell, unlike reported by Müller et al. (2008), or the sodium silicate has a detrimental effect on growth.

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Discussion & Conclusions

In this part of our project we have transformed E. coli with a gene encoding the enzyme silicatein in three different ways. We solely expressed the silicatein protein, but have also fused the protein to two different membrane proteins, INP and OmpA. The goal was to let the cells produce a polysilicate layer around their membrane, so the cell was completely covered in this biological glass. We have characterized the polysilicate-covered cells by electron microscopy, AFM, fluorescent staining and with a growth study.

After staining the silicatein-expressing cells with the fluorescent stain Rhodamine 123, which binds specifically to the polysilicate layer, we found that the cells transformed with the OmpA-silicatein fusion successfully synthesized a polysilicate layer around the cell, therefor we continued all experiments with this strain. This result was confirmed with TEM and EDX spectroscopy, where a higher concentration of silicon was found on the polysilicate-synthesizing cells. SEM showed that this layer is a neat, homogeneous layer and does not form any aggregates of polysilicate on the cell. Aggregates could seriously disturb the optical properties of our cell, so it’s a good results that our cells are covered homogeneously. AFM showed that the cells covered in polysilicate have a higher stiffness sompared to cells without the polysilicate layer. This proves the presence of this layer. The finding that the cells are more rigid is also useful for our idea to develop biolenses, since these should not easily break. Lastly, our growth study shows that the cells covered in polysilicate live shorter that cells without this layer. This means that when we want to produce microlenses at a high scale, we should first grow the cells prior to incubating them with silicic acid. The fact that the cells die when they are encapsulated could also make it easier to use our microlenses in user-products. Bringing GMO’s out of the lab is often not permitted, so if we can show that our biolenses are inviable, this would not be a problem for our product.

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 home-built 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:

Setup results — **Figure 2,** *E. coli* cells transformed with (A)OmpA-silicatein fusion plasmid and (B) mCerulean, imaged in our custom self-built optical setup. The cells were excited with a laser at a wavelength of 405nm and an intensity of 0.5 mW.

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.

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Intensity measurements of laser cells

Introduction

One of the biggest differences between a laser and fluorescence is the amount of emitted photons. We can measure the amount of emitted photons by measuring the intensity of emitted light. The intensity of fluorescence increases linearly with the excitation energy. However, at a certain excitation energy it will reach the so-called ‘laser threshold’ and stimulated emission, and thus lasing will occur. From this point on, the output intensity of the fluorophores will still increase linearly, but with a much steeper slope, as shown in figure 1.

Fluorescence vs. lasing — **Figure 1,** the relation of input energy vs. output intensity for fluorescence and lasing (Fan & Yun, 2014)

We investigated whether we could find the same relation between input and output intensity for our Biolaser cells, using our self-built setup, to investigate whether our cells were able

Methods

For this experiment we used E. coli BL21 transformed with the following BioBricks:

Plasmid(s) and conditions	Function
mCerulean (constitutively expressed)	Fluorescence
mCerulean (constitutive) + OmpA-silicatein (induced, incubated in silicic acid)	Biolaser
OmpA-silicatein (induced, incubated in silicic acid)	Negative control (no fluorescence)

The cells were fixated on a microscope slide using 3% agarose pads. The cells were excited at a wavelength of 405 nm. The cells were imaged at excitation energies of 0.1 mW, 0.5 mW, 0.7 mW, 1 mW, 2 mW, 5 mW, 10 mW and 50 mW. These images were analysed using ImageJ to determine the output intensity and corrected for background noise (McCloy et al., 2014).

Results and discussion

The output intensities of our cells were plotted against the excitation power to determine whether our cells emitted laser-like light. The results are shown in figure 2.

In figure 2, the measured intensity is plotted against the excitation power (black dots). Since we expect a linear relation, the expected increase of fluorescence was also plotted (in a blue line). If the slope of the measurement data is higher than this slope, we observe lasing. If the slope is lower than the expected line, we observe photobleaching. In figure 2 we see that the measurement data first nicely matches the expected increase in fluorescence. However, at an excitation power of 2 mW we see that in all cases the fluorescence stays at the same level. This means that the intensity is not increasing anymore and we observe photobleaching. So, both the fluorescent cells as well as the laser cells do not emit any laser-like light. For the fluorescent cells that were transformed with solely mCerulean, this result is as expected, these cells do not have any extra modifications that should cause them to emit laser light. The strain that was transformed with both mCerulean and OmpA-silicatein had a glass shell around the cell that could cause the cells to emit laser light. This effect was not observed. The strain transformed with solely OmpA-silicatein did not emit any light (results not shown).

From this experiment we can conclude that the cells were not able to emit laser light. This could be both due tour setup or due to the constructed cells. The lasing should have occurred at an excitation power under 1mW (Fan & Yun, 2014). However, up to this excitation power the Biolaser cells follow the same slope as the fluorescent cells, which means that no lasing occurred. From 2 mW and higher the cells bleach. Since we expected the cells to lase at an excitation energy under 1 mW, we did not take such high energies into account while designing our setup. Even though we exposed the cells to the excitation laser for a very short time, the power was so high that the cells eventually photobleached anyway.

Even though the excitation laser eventually bleached the fluorophores, this was probably not the reason why lasing didn’t work. Lasing should have occurred at an excitation energy between 0 and 1 mW, and at these energies the fluorophores did not bleach. This indicated that there could be another reason why the cells did not lase. Modeling showed, that in a cavity as big as E. coli (around 1 µm) we need an intracellular fluorophore concentration of 0.1 M to get lasing. In our cells the maximum concentration we can achieve is in the nM to µM range and therefore lasing physically not possible in our cells. To get lasing, we would either need much larger cells or a much higher concentration of fluorophores. Therefore, it was most likely not due to the self-built setup that we did not observe lasing.

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Discussion & Conclusions

We have successfully calculated, designed and built a custom optical setup that could allow us to measure lasing in cells. Using this self-built setup, we were able to image fluorescent cells and measure the intensity of the fluorescence. We have confirmed that the setup worked. However, we did not observe any lasing in cells. Our models have shown that this is due to the size of our cells, if we would use a bigger organism, e.g. a mammalian cell line, we could be able to measure lasing. Unfortunately, we were not able to measure this in our setup, since we did not have the safety permit to use mammalian cells. However, this would be a very interesting future study.

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.

Diffraction — **Figure 1:** Different ways of focusing light by rod-shaped lenses and spherical lenses. The rod-shaped lens has various directions in which it can break the light, therefore the orientation of this lens is extremely important. The spherical cell only has one, so the orientation of the cell does not matter.

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). We will express this gene both under a constitutive promoter (Part K1890031), as well as an inducible promoter (Part K1890030). 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.

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Sterilization of the biolenses

Introduction

We wanted to test our biolenses in a real-world application: solar cells. This was chosen, since it was shown to make solar cells more efficient while using microlenses (Jutteau et al., 2015). To do these experiments, Dr. Arno Smets, professor Photovoltaic Materials and Devices of the TU Delft had sent us to Stefaan Heirman, a technician at the same research group, who helped us. However, our biolenses should leave the ML-1 lab, as Stefaan was located at another faculty in Delft. If in the future the biolenses will be used on solar panels, they are in the environment as well. Since we want to be able to implement our biolenses in a real-world application, the biolenses needed to be sterilized.

To sterilize, different options are available. For example alcohols, chlorine and chlorine compounds are used within healthcare and autoclaving is a much-used method in laboratory settings. Besides that, UV sterilization is a much-used method in water treatment (Chang et al., 1985; Hijnen et al., 2006; O’Flaherty et al., 2016). This is used to remove among others E. coli. Since we do not want any chemicals to potentially interfere with our biolenses, we have chosen not to use any of these chemicals and to test whether autoclaving or UV sterilization would be a good option.

Methods

Autoclaving biolenses

Aliquots of cells were autoclaved before imaged with scanning electron microscopy (SEM).

UV sterilization

A volume of 30 µL E. coli BL21 cells that were transformed with BolA in pBbA5c was plated on LB plates complemented with chloramphenicol. As controls for sterilization aliquots of 500 µL of the same cells were either heated at 95 °C for 10 minutes or washed with 500 µL 70% ethanol for three times. After sterilization, also 30 µL of these cells were plated on LB+Cm plates. Besides that, 30 µL of non sterilized cells were plated as well.

Two of the plates containing BolA transformed cells were UV sterilized in a Stratagene UV Stratalinker 1800. The manufacturer recommended power settings of 120000 µJ. In our experiment we have used a power of 120000 µJ and 240000µJ to sterilize our cells.

All plates were incubated overnight at 37 °C.

Results

Autoclaving

Cells containing the Sil_Sdom gene were autoclaved and subsequently imaged with SEM.

Figure 1. *E. coli* BL21 cells transformed with Sil_Sdom, provided with silicic acid and were autoclaved and subsequently imaged by with SEM.

Since the SEM showed the cells were collapsed, and in no way could function as biolenses, another sterilization method was used.

UV sterilization

To test the effectivity of UV sterilization on our E. coli cells, a growth study after UV sterilization was performed. For this 30 µL BL21 cells transformed with BolA in pBbA5c was plated on LB plates complemented with chloramphenicol. These plates were UV sterilized in a Strategene UV Stratalinker 1800. As positive controls the same cells were heated for 10 minutes at 95 ºC or washed three times with 500 µL ethanol. As negative control the same cells were plated on a selective plate.

None of the sterilized plates showed any growth, while the non sterilized plate did yield colonies.

Besides the growth study, the cells were also imaged with SEM, which can be found in figure 2.

Figure 2. *E. coli* BL21 cells transformed with Sil_Sdom, provided with silicic acid and were UV sterilized and subsequently imaged by with SEM.

As shown in figure 2, the with silicate capturing of the cells remain undamaged after UV sterilization and could be used as biolenses now.

Discussion

In this experiment, different methods to sterilize our biolenses were tested, as we wanted to be able to put our biological microlenses in real-world conditions. For this, we have both autoclaved and UV sterilized our cells. From the growth study, it can be concluded that the biolenses were successfully sterilized by UV sterilization. Besides that, the shape of the biolenses was not affected by UV sterilization as opposed to autoclaving.

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Improving solar cells with biolenses

Introduction

Ways to measure the efficiency of photovoltaics

There are different ways to measure the output of photovoltaic (PV) cells with accuracy and repeatability. The most used characteristic of PVs is their efficiency (Power Conversion Efficiency or PCE). It is defined as the energy produced from the device by the intended radiation from a light source. In order to measure the efficiency and produce acceptable results; a set protocols were set in the scientific community.

Those protocols are relevant for the intended radiation and the measurements taken. For the intended radiation we make the assumptions of air mass 1.5 (dictates how the sun’s radiation is influenced by the atmosphere), the measured surface is 37 degrees from the equator and 48.2 degrees from Zenith. Finally, the radiation is 1000Wm^-2 (Snaith, 2012) .

The parameters that influence the PCE measurements can be found in characteristic J-V diagrams, plotting current density (A/cm2) against voltage (V) (figure 1). The graph contains the three basic parameters of PCE measurements. Those are the short circuit current (I_sc), the open circuit voltage (V_oc) and the Fill Factor (FF). This graph is obtained by scanning the photovoltaic’s current output within a range of voltages, usually from -1 Volts to +1 Volts. The V_oc is the voltage when the current is zero and the I_sc is the current when the voltage is zero. The Fill Factor represents how much power the solar cell is producing or its theoretical maximum. The theoretical maximum power for each solar cell is \( P_{ideal} = V_{oc} \times I_{sc} \) and the maximum power is \( P_{max} = V_{max} \times I_{max}\) then the Fill Factor is defined as \(FF = \frac{P_{max}}{P_{ideal}} \). The Fill Factor is a defining term of the behavior of the solar cells (Grossiord, Kroon, Andriessen, & Blom, 2012).

Fill Factor of PV devices — Figure 1. Characteristic curve of PV measurements.

Equipment and proposed measurements

In order to achieve those reproducible and accurate measurement of efficiency a certain type of equipment complemented with previously mentioned protocols need to be used. The equipment used to simulate the aforementioned conditions is called Solar Simulator, which is basically a big Xenon lamp with well-defined light power output. In order to calibrate the simulator a ‘standard’ cell is used with well-defined properties to measure the exact output of the simulator in each measurement. Finally, the tested device must be placed perpendicular to the light with nothing restricting the light path and not moved during the whole testing. Using this set of equipment, we are sure that we conduct all the experiments under the same scientifically accepted conditions so we remove a factor of uncertainty to our experimental results.

Methods

Results

Discussion

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Discussion & Conclusions

Conclusion and discussion on the experiments

Conclusions & Recommendations

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.
Cha, J. N., Shimizu, K., Zhou, Y., Christiansen, S. C., Chmelka, B. F., Stucky, G. D., & Morse, D. E. (1999). Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proceedings of the National Academy of Sciences, 96(2), 361-365.
Chang, J. C., Ossoff, S. F., Lobe, D. C., Dorfman, M. H., Dumais, C. M., Qualls, R. G., & Johnson, J. D. (1985). UV inactivation of pathogenic and indicator microorganisms. Applied and environmental microbiology, 49(6), 1361-1365.
Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.
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-a Journal of the American Chemical Society, 127(45), 15749–15755.
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.
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.
Electron microscopy. (2013). Slideshare.net. Retrieved 15 October 2016, from http://www.slideshare.net/suniu/electron-microscopy
Fan, X., & Yun, S.-H. (2014). The potential of optofluidic biolasers. Nature methods, 11(2), 141-147.
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.
Friel, J. J., & Lyman, C. E. (2006). Tutorial review: X-ray mapping in electron-beam instruments. Microscopy and Microanalysis, 12(01), 2-25.
Gather, M. C., & Yun, S. H. (2011). Single-cell biological lasers. Nature Photonics, 5(7), 406-410.
Grossiord, N., Kroon, J. M., Andriessen, R., & Blom, P. W. M. (2012). Degradation mechanisms in organic photovoltaic devices. Organic Electronics, 13(3), 432–456. http://doi.org/10.1016/j.orgel.2011.11.027
Hardy, J., & Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297(5580), 353-356.
Heintzmann, R., & Ficz, G. (2006). Breaking the resolution limit in light microscopy. Briefings in functional genomics & proteomics, 5(4), 289-301.
Hijnen, W. A. M., Beerendonk, E. F., & Medema, G. J. (2006). Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo) cysts in water: a review. Water research, 40(1), 3-22.
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.
Jutteau, S., Paire, M., Proise, F., Lombez, L., & Guillemoles, J. F. (2015, 14-19 June 2015). Micro solar concentrators: Design and fabrication for microcells arrays. Paper presented at the Photovoltaic Specialist Conference (PVSC), 2015 IEEE 42nd
Kim, E.-J., & Yoo, S.-K. (1998). Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein. Biotechnology techniques, 12(3), 197-201.
Kwakernaak, I. C., & Sloof, I. W. G. (2005). Introduction in the use and application of Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy in materials., (November), 1–14.
Lakowicz, J. R. (2013). Principles of fluorescence spectroscopy: Springer Science & Business Media.
Li, C.-W., Chu, S., & Lee, M. (1989). Characterizing the silica deposition vesicle of diatoms. Protoplasma, 151(2-3), 158-163.
McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., & Burgess, A. (2014). Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events. Cell Cycle, 13(9), 1400-1412.
Müller, W. E. G. (2003). Silicon biomineralization.
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.
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.
Müller, W. E. (2011). Molecular biomineralization: aquatic organisms forming extraordinary materials: Springer Science & Business Media.
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).
Nam, M., Kim, K., Lee, J., Yang, S. S., & Lee, K.-K. (2013). Solar cell packaged by a microlens array and its long-term optical efficiency enhancement. Paper presented at the Proc. of SPIE Vol.
O’Flaherty, E., Membré, J. M., & Cummins, E. (2016). META-ANALYSIS OF THE EFFECT OF UV WATER TREATMENT ON ESCHERICHIA COLI. BIOSYSTEMS AND FOOD ENGINEERING RESEARCH REVIEW 21, 147.
Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).
Nellist, P. D. (2011). The principles of STEM imaging. In Scanning Transmission Electron Microscopy (pp. 91-115). Springer New York.
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.
Shcherbo, D., Merzlyak, E. M., Chepurnykh, T. V, Fradkov, A. F., Ermakova, G. V, Solovieva, E. a, … Chudakov, D. M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nature Methods, 4(9), 741–746. http://doi.org/10.1038/nmeth1083
Snaith, H. J. (2012). The perils of solar cell efficiency measurements, 6(June), 337–340.
Svelto, O., & Hanna, D. C. (1976). Principles of lasers: Springer.

                                           
    
    

        
            
        
        
            
        


        
        

            

            


            

                
                    

                        

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[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] Cha, J. N., Shimizu, K., Zhou, Y., Christiansen, S. C., Chmelka, B. F., Stucky, G. D., & Morse, D. E. (1999). Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proceedings of the National Academy of Sciences, 96(2), 361-365.

[3] Chang, J. C., Ossoff, S. F., Lobe, D. C., Dorfman, M. H., Dumais, C. M., Qualls, R. G., & Johnson, J. D. (1985). UV inactivation of pathogenic and indicator microorganisms. Applied and environmental microbiology, 49(6), 1361-1365.

[4] Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.

[5] 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-a Journal of the American Chemical Society, 127(45), 15749–15755.

[6] 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.

[7] 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.

[8] Electron microscopy. (2013). Slideshare.net. Retrieved 15 October 2016, from http://www.slideshare.net/suniu/electron-microscopy

[9] Fan, X., & Yun, S.-H. (2014). The potential of optofluidic biolasers. Nature methods, 11(2), 141-147.

[10] 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.

[11] Friel, J. J., & Lyman, C. E. (2006). Tutorial review: X-ray mapping in electron-beam instruments. Microscopy and Microanalysis, 12(01), 2-25.

[12] Gather, M. C., & Yun, S. H. (2011). Single-cell biological lasers. Nature Photonics, 5(7), 406-410.

[13] Grossiord, N., Kroon, J. M., Andriessen, R., & Blom, P. W. M. (2012). Degradation mechanisms in organic photovoltaic devices. Organic Electronics, 13(3), 432–456. http://doi.org/10.1016/j.orgel.2011.11.027

[14] Hardy, J., & Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297(5580), 353-356.

[15] Heintzmann, R., & Ficz, G. (2006). Breaking the resolution limit in light microscopy. Briefings in functional genomics & proteomics, 5(4), 289-301.

[16] Hijnen, W. A. M., Beerendonk, E. F., & Medema, G. J. (2006). Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo) cysts in water: a review. Water research, 40(1), 3-22.

[17] 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.

[18] 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.

[19] Jutteau, S., Paire, M., Proise, F., Lombez, L., & Guillemoles, J. F. (2015, 14-19 June 2015). Micro solar concentrators: Design and fabrication for microcells arrays. Paper presented at the Photovoltaic Specialist Conference (PVSC), 2015 IEEE 42nd

[20] Kim, E.-J., & Yoo, S.-K. (1998). Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein. Biotechnology techniques, 12(3), 197-201.

[21] Kwakernaak, I. C., & Sloof, I. W. G. (2005). Introduction in the use and application of Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy in materials., (November), 1–14.

[22] Lakowicz, J. R. (2013). Principles of fluorescence spectroscopy: Springer Science & Business Media.

[23] Li, C.-W., Chu, S., & Lee, M. (1989). Characterizing the silica deposition vesicle of diatoms. Protoplasma, 151(2-3), 158-163.

[24] McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., & Burgess, A. (2014). Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events. Cell Cycle, 13(9), 1400-1412.

[25] Müller, W. E. G. (2003). Silicon biomineralization.

[26] 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.

[27] 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.

[28] Müller, W. E. (2011). Molecular biomineralization: aquatic organisms forming extraordinary materials: Springer Science & Business Media.

[29] Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

[30] Nam, M., Kim, K., Lee, J., Yang, S. S., & Lee, K.-K. (2013). Solar cell packaged by a microlens array and its long-term optical efficiency enhancement. Paper presented at the Proc. of SPIE Vol.

[31] O’Flaherty, E., Membré, J. M., & Cummins, E. (2016). META-ANALYSIS OF THE EFFECT OF UV WATER TREATMENT ON ESCHERICHIA COLI. BIOSYSTEMS AND FOOD ENGINEERING RESEARCH REVIEW 21, 147.

[32] Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

[33] Nellist, P. D. (2011). The principles of STEM imaging. In Scanning Transmission Electron Microscopy (pp. 91-115). Springer New York.

[34] 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.

[35] Shcherbo, D., Merzlyak, E. M., Chepurnykh, T. V, Fradkov, A. F., Ermakova, G. V, Solovieva, E. a, … Chudakov, D. M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nature Methods, 4(9), 741–746. http://doi.org/10.1038/nmeth1083

[36] Snaith, H. J. (2012). The perils of solar cell efficiency measurements, 6(June), 337–340.

[37] Svelto, O., & Hanna, D. C. (1976). Principles of lasers: Springer.

@@ Line 196: / Line 196: @@
                      <p>Since mVenus, mKate and mCerulean did not exist in the iGEM registry yet, we
                          constructed a brand new part for each of these fluorophores including strong constitutive
-                         promoter and RBS. GFP, on the other hand, was present in a whole
+                         promoter and RBS (parts <b><a href="http://parts.igem.org/Part:BBa_K1890010" target="_blank">K1890010</a></b>
+                        and <b><a href="http://parts.igem.org/Part:BBa_K1890011" target="_blank">K1890011</a></b>).
+                        GFP, on the other hand, was present in a whole
                          range of biobricks. However, to our knowledge, there was no single biobrick
                          available containing promoter, RBS and terminators. Hence, we constructed a
@@ Line 207: / Line 209: @@
                          <b><a href="http://parts.igem.org/Part:BBa_J23105" target="_blank">J23105</a></b>, and <b><a href="http://parts.igem.org/Part:BBa_J23108" target="_blank">J23108</a></b>.
                          This way we were able to see the influence of promoter strength on
-                         fluorescent output.</p>
+                         fluorescent output (parts <b><a href="http://parts.igem.org/Part:BBa_K1890020" target="_blank">K1890020</a></b>
+                        to <b><a href="http://parts.igem.org/Part:BBa_K1890024" target="_blank">K1890024</a></b>.</p>
@@ Line 238: / Line 241: @@
                                      <p>To assure the fluorophores were functional, the emission spectra were recorded at the given excitation wavelength.</p>
                                      <h3>Methods</h3>
-                                     <p>Parts encoding the four different fluorophores, including promoter, RBS and terminators, were expressed in <i>E. coli</i> 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. </p>
+                                     <p>Parts encoding the four different fluorophores, including promoter, RBS and terminators, were expressed in <i>E. coli</i> BL21.
-                                     <p>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.  </p>
+                                        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. </p>
+                                     <p>All of the mentioned biobricks were expressed in <i>E. coli</i> BL21.
+                                        After growing in LB medium to an OD600 of ~1, the cells were washed and resuspended in the same volume of PBS. Aliquots of 100 µL were put in a 96 well plate.  </p>
                                      <p>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.
                                          This can be seen in the figures, where the left half of the emission peaks could not be measured. Especially for mVenus, where the excitation and emission wavelengths are very close together.</p>
@@ Line 255: / Line 262: @@
                                              <figcaption><b>Figure 2,</b> Emission spectra of GFP expressed under control of promoters with different strengths. Excitation wavelength was 488 nm.</figcaption></center>
                                      </figure>
-                                     <p>For comparison, the emission was normalized by dividing by OD. All strains were measured in the same dilution, in order to make the results reproducible. The emission intensity is as expected, according to the promoter strength. All fluorophore spectra were also recorded in a dilution more suited for their emission intensity and normalized by 1 (Figure 3). From this figure we can conclude that all GFP biobricks function properly.</p>
+                                     <p>For comparison, the emission was normalized by dividing by OD. All strains were measured in the same dilution, in order to make the results reproducible. The emission intensity is as expected, corresponding to the strength of the promoters. All fluorophore spectra were also recorded in a dilution more suited for their emission intensity and normalized by 1 (Figure 3). From this figure we can conclude that all GFP biobricks function properly.</p>
                                      <figure>
                                          <center><img src="https://static.igem.org/mediawiki/2016/4/46/T--TU_Delft--Spectra_GFP_individual.png" alt="fluorophores">
@@ Line 291: / Line 298: @@
                                      <h3>Methods</h3>
                                      <p>
-                                         An overnight culture in eM9 medium was inoculated in fresh eM9 to an OD of 0.1
+                                         An overnight culture in eM9 medium was inoculated in fresh eM9 to an OD600 of 0.1
                                          in a 96 well plate. The emission at 522 nm was measured every 15 minutes.
                                          Measurements were done in quadruplicate with pure eM9 as a blank.
@@ Line 298: / Line 305: @@
                                      <p>
                                          Figure 1 shows the 24 hour measurement of optical density and fluorescence
-                                         intensity. The final OD is approximately equal for all different strains,
+                                         intensity. The final OD600 is approximately equal for all different strains,
                                          suggesting that the level of constitutive expression was not influencing the
                                          growth. Furthermore, fluorescence intensity drops after the exponential growth
@@ Line 314: / Line 321: @@
                                      <p>
                                          Cells expressing mCerulean or mVenus, however, seem to be having a longer lag phase before
-                                         exponential growth starts (Figure 2). Also, they reach a lower final OD than the ones expressing
+                                         exponential growth starts (Figure 2). Also, they reach a lower final OD600 than the ones expressing
                                          GFP. These fluorophores might be slightly harmfull for cell growth. Nonetheless, they do grow
                                          and exhibit fluorescence, so they can be used in further experiments.
@@ Line 341: / Line 348: @@
                          for the iGEM registry: <b><a href="http://parts.igem.org/Part:BBa_K1890011" target="_blank">mVenus</a></b>
                          and <b><a href="http://parts.igem.org/Part:BBa_K1890010" target="_blank">mCerulean</a></b>.
-                         Also, we constructed <b><a href="http://parts.igem.org/Part:BBa_K1890020" target="_blank">five new composite parts</a></b>,
+                         Both of them exhibit a bright fluorescence, which is useful in our futher experiments.
-                         based on the existing GFP biobrick. All in all, these new parts provide a ready-to-go expression device for green, yellow, or cyan fluorescence,
+                        To our knowledge there was no part available in the registry consisting of promoter, RBS,
+                        GFP coding sequence and terminator Therefore, we constructed
+                        <b><a href="http://parts.igem.org/Part:BBa_K1890020" target="_blank">five new composite parts</a></b>
+                         to provide single complete biobricks for GFP expression. These new parts were characterized and show
+                        a bright fluorescence, according to their promoter strength.
+                        All in all, our new parts provide a ready-to-go expression device for green, yellow, or cyan fluorescence,
                          which can be very usefull for future iGEM teams.</p><br>
@@ Line 488: / Line 500: @@
                                  <div class="panel-body">
-<h3>Introduction</h3>
+                                    <h3>Introduction</h3>
                                      <p>Electron microscopy is an imaging technique that can acquire images with resolutions
                                          far above the optical limit due to the small wavelength of electrons <a href="#references"> (Nellist, 2011)</a>.
@@ Line 509: / Line 521: @@
                                          specifically determine which elements are present in our sample. </p>
                                      <h3> Methods</h3>
-                                     <p>The experiment were performed using <i> E.coli</i> BL21 cells
+                                     <p>The experiment were performed using <i> <i>E. coli</i></i> BL21 cells
                                          with the plasmid containing OmpA-Silicatein. Two samples were made
                                          where the first sample was induced with IPTG but no silicic acid was added and a second sample which was both induced with IPTG and silicic acid was added, therefore
@@ Line 551: / Line 563: @@
                              <div id="collapseDos" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingDos">
                                  <div class="panel-body">
-                                   <h3>Introduction</h3>
+                                    <h3>Introduction</h3>
-<p>The Scanning Electron Microscope, or SEM as it is called, it is one of the most widely used imaging techniques in science. It is highly used due to its very high spatial resolution and Depth of Focus (DoF). SEM can reach resolution of 5 nm and DoF about 0.5 mm, compared to an optical microscope that can reach a spatial resolution of \( 0.2 \mu m \) and DoF \( 1 \mu m \). Important here is to note that with SEM it is only possible to image solid samples because the measurements take place under vacuum. Since we want to image the impact of the polysilicate layer in the highest detail, we use sem to image our samples.</p>
+                                    <p>The Scanning Electron Microscope, or SEM as it is called, it is one of the most widely used imaging techniques in science. It is highly used due to its very high spatial resolution and Depth of Focus (DoF). SEM can reach resolution of 5 nm and DoF about 0.5 mm, compared to an optical microscope that can reach a spatial resolution of \( 0.2 \mu m \) and DoF \( 1 \mu m \). Important here is to note that with SEM it is only possible to image solid samples because the measurements take place under vacuum. Since we want to image the impact of the polysilicate layer in the highest detail, we use sem to image our samples.</p>
-<p>In SEM a beam of electrons is accelerated by high voltage and it is directed towards the sample with speeds up to \( 1.64 \times 10^5 km\). When the beam hits the sample, x-rays, backscattered electrons and secondary electrons are generated. For imaging, SEM uses the secondary electrons. Those secondary electrons are then collected and their kinetic energy is transformed to an image. <a href="#references">(Kwakernaak & Sloof, 2005)</a></p>
+                                    <p>In SEM a beam of electrons is accelerated by high voltage and it is directed towards the sample with speeds up to \( 1.64 \times 10^5 km\). When the beam hits the sample, x-rays, backscattered electrons and secondary electrons are generated. For imaging, SEM uses the secondary electrons. Those secondary electrons are then collected and their kinetic energy is transformed to an image. <a href="#references">(Kwakernaak & Sloof, 2005)</a></p>
@@ Line 601: / Line 613: @@
                                  <div class="panel-body">
-<h3>Introduction</h3>
+                                    <h3>Introduction</h3>
                                      <p>Due to encapsulation of the cell with a layer of polysilica the stiffness could change.
@@ Line 620: / Line 632: @@
                                      </figure>
                                      <h3> Methods</h3>
-                                     <p> Experiments were performed using <i> E.coli </i> BL21 cells transformed with the plasmid containing the OmpA-Silicatein
+                                     <p> Experiments were performed using <i> <i>E. coli</i> </i> BL21 cells transformed with the plasmid containing the OmpA-Silicatein
                                          gene. Silicic acid was added to one sample to form the polysilicate layer around the cell. Another sample with no silicic acid added was
                                          used as a control. The cells weres spun down and resuspended in MilliQ and fixated on a glass slide using 1% ABTES. </p>
@@ Line 632: / Line 644: @@
                                      <figure>
                                          <img src = "https://static.igem.org/mediawiki/2016/thumb/f/f5/T--TU_Delft--AFM_Data.png/783px-T--TU_Delft--AFM_Data.png" alt= " ">
-                                         <figcaption> <b>Figure 2:</b> Pictures taken with AFM of (A-B) <i>E.coli</i> transformed with OmpA-fused silicatein
+                                         <figcaption> <b>Figure 2:</b> Pictures taken with AFM of (A-B) <i>E. coli</i> transformed with OmpA-fused silicatein
-                                             with silicic acid added, (C-D) <i>E.coli</i> transformed with OmpA fused to silicatein without silic acid added.
+                                             with silicic acid added, (C-D) <i>E. coli</i> transformed with OmpA fused to silicatein without silic acid added.
                                              (A,C) are height maps of the cell, (B,D) are stiffness maps.
                                          </figcaption>
@@ Line 644: / Line 656: @@
                                      <figure>
                                          <img src = "https://static.igem.org/mediawiki/2016/d/db/T--TU_Delft--AFMRelativeStiffness.png" alt= " ">
-                                         <figcaption> <b>Figure 3:</b> Relative stiffness of <i>E.coli</i> cells covered with and without polysilicate layer, compared to the stiffness of a glass slide
+                                         <figcaption> <b>Figure 3:</b> Relative stiffness of <i>E. coli</i> cells covered with and without polysilicate layer, compared to the stiffness of a glass slide
                                              measured with Peakforce QNM AFM.  </figcaption>
                                      </figure>
@@ Line 684: / Line 696: @@
                                              <figcaption><b>Figure 1,</b> Number of colony forming units (cfu) during incubation with silicic acid.</figcaption></center>
                                      </figure>
-<p>This figure suggests that either the polysilicate layer inhibits nutrient diffusion
+                                    <p>This figure suggests that either the polysilicate layer inhibits nutrient diffusion
-into the cell, unlike reported by Müller et al. (2008), or the sodium silicate has a detrimental
+                                        into the cell, unlike reported by Müller et al. (2008), or the sodium silicate has a detrimental
-effect on growth.</p>
+                                        effect on growth.</p>
                                      <a href="#silicatein" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#silicatein').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
@@ Line 699: / Line 711: @@
                      <p>In this part of our project we have transformed <i>E. coli</i> with a gene encoding the enzyme silicatein in three different ways. We solely expressed the silicatein protein, but have also fused the protein to two different membrane proteins, INP and OmpA. The goal was to let the cells produce a polysilicate layer around their membrane, so the cell was completely covered in this biological glass. We have characterized the polysilicate-covered cells by electron microscopy, AFM, fluorescent staining and with a growth study.</p>
-<p>After staining the silicatein-expressing cells with  the fluorescent stain Rhodamine 123, which binds specifically to the polysilicate layer, we found that the cells transformed with the OmpA-silicatein fusion successfully synthesized a polysilicate layer around the cell, therefor we continued all experiments with this strain. This result was confirmed with TEM and EDX spectroscopy, where a higher concentration of silicon was found on the polysilicate-synthesizing cells. SEM showed that this layer is a neat, homogeneous layer and does not form any aggregates of polysilicate on the cell. Aggregates could seriously disturb the optical properties of our cell, so it’s a good results that our cells are covered homogeneously. AFM showed that the cells covered in polysilicate have a higher stiffness sompared to cells without the polysilicate layer. This proves the presence of this layer. The finding that the cells are more rigid is also useful for our idea to develop biolenses, since these should not easily break. Lastly, our growth study shows that the cells covered in polysilicate live shorter that cells without this layer. This means that when we want to produce microlenses at a high scale, we should first grow the cells prior to incubating them with silicic acid. The fact that the cells die when they are encapsulated could also make it easier to use our microlenses in user-products. Bringing GMO’s out of the lab is often not permitted, so if we can show that our biolenses are inviable, this would not be a problem for our product.</p>
+                    <p>After staining the silicatein-expressing cells with  the fluorescent stain Rhodamine 123, which binds specifically to the polysilicate layer, we found that the cells transformed with the OmpA-silicatein fusion successfully synthesized a polysilicate layer around the cell, therefor we continued all experiments with this strain. This result was confirmed with TEM and EDX spectroscopy, where a higher concentration of silicon was found on the polysilicate-synthesizing cells. SEM showed that this layer is a neat, homogeneous layer and does not form any aggregates of polysilicate on the cell. Aggregates could seriously disturb the optical properties of our cell, so it’s a good results that our cells are covered homogeneously. AFM showed that the cells covered in polysilicate have a higher stiffness sompared to cells without the polysilicate layer. This proves the presence of this layer. The finding that the cells are more rigid is also useful for our idea to develop biolenses, since these should not easily break. Lastly, our growth study shows that the cells covered in polysilicate live shorter that cells without this layer. This means that when we want to produce microlenses at a high scale, we should first grow the cells prior to incubating them with silicic acid. The fact that the cells die when they are encapsulated could also make it easier to use our microlenses in user-products. Bringing GMO’s out of the lab is often not permitted, so if we can show that our biolenses are inviable, this would not be a problem for our product.</p>
@@ Line 758: / Line 770: @@
                                      <p>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.</p>
                                      <h3>Methods</h3>
-                                     <p>In order to confirm whether the setup was working, we used <i> E. coli</i> 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.</p>
+                                     <p>In order to confirm whether the setup was working, we used <i> <i>E. coli</i></i> 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.</p>
                                      <p>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.</p>
                                      <h3>Results & discussion</h3>
@@ Line 764: / Line 776: @@
                                      <figure>
                                          <img src="https://static.igem.org/mediawiki/2016/2/2a/T--TU_Delft--Cellsinsetup.png" alt="Setup results"  >
-                                         <figcaption> <b>Figure 2,</b> <i> E. coli</i> cells transformed with (A)OmpA-silicatein fusion plasmid and (B) mCerulean, imaged in our custom self-built optical setup. The cells were excited with a laser at a wavelength of 405nm and an intensity of 0.5 mW. </figcaption>
+                                         <figcaption> <b>Figure 2,</b> <i> <i>E. coli</i></i> cells transformed with (A)OmpA-silicatein fusion plasmid and (B) mCerulean, imaged in our custom self-built optical setup. The cells were excited with a laser at a wavelength of 405nm and an intensity of 0.5 mW. </figcaption>
                                      </figure>
                                      <p>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. </p>
@@ Line 919: / Line 931: @@
                                          advantage of being symmetrical along all axes, so the orientation of your lens does not
                                          matter for the optical properties.</p>
-                                     <p>In order to create spherical E. coli, we overexpress the <i>BolA</i> gene.
+                                     <p>In order to create spherical <i>E. coli</i>, we overexpress the <i>BolA</i> gene.
                                          <i>BolA</i> is a gene that controls the morphology of <i>E. coli</i> in the stress
                                          response <a href="#references">(Santos, Freire, Vicente, & Arraiano, 1999)</a>.
@@ Line 968: / Line 980: @@
                                      <figure><center>
                                              <img src="https://static.igem.org/mediawiki/2016/e/e6/T--TU_Delft--BolA_widefield.png" alt="BolA">
-                                             <figcaption><b> Figure 2: </b> Widefield images of <i>E.coli</i> BL21 transformed with <b>(A)</b> <i>BolA</i>
+                                             <figcaption><b> Figure 2: </b> Widefield images of <i>E. coli</i> BL21 transformed with <b>(A)</b> <i>BolA</i>
                                                  under the inducible Lac-promoter, uninduced, <b>(B)</b> <i>BolA</i> under the inducible Lac promoter and
                                                  induced with 1 mM IPTG, <b>(C)</b> <i>BolA</i> under the constitutive promoter J23100, <b>(D)</b>
@@ Line 1,020: / Line 1,032: @@
                                      <h3>Introduction<h3>
-<p>We wanted to test our biolenses in a real-world application: solar cells. This was chosen, since it was shown to make solar cells more efficient while using microlenses <a href="#references">(Jutteau <i>et al.</i>, 2015)</a>. To do these experiments, Dr. Arno Smets, professor Photovoltaic Materials and Devices of the TU Delft had sent us to Stefaan Heirman, a technician at the same research group, who helped us. However, our biolenses should leave the ML-1 lab, as Stefaan was located at another faculty in Delft. If in the future the biolenses will be used on solar panels, they are in the environment as well. Since we want to be able to implement our biolenses in a real-world application, the biolenses needed to be sterilized. </p>
+                                            <p>We wanted to test our biolenses in a real-world application: solar cells. This was chosen, since it was shown to make solar cells more efficient while using microlenses <a href="#references">(Jutteau <i>et al.</i>, 2015)</a>. To do these experiments, Dr. Arno Smets, professor Photovoltaic Materials and Devices of the TU Delft had sent us to Stefaan Heirman, a technician at the same research group, who helped us. However, our biolenses should leave the ML-1 lab, as Stefaan was located at another faculty in Delft. If in the future the biolenses will be used on solar panels, they are in the environment as well. Since we want to be able to implement our biolenses in a real-world application, the biolenses needed to be sterilized. </p>
-<p>To sterilize, different options are available. For example alcohols, chlorine and chlorine compounds are used within healthcare and autoclaving is a much-used method in laboratory settings. Besides that, UV sterilization is a much-used method in water treatment <a href="#references">(Chang <i>et al.</i>, 1985; Hijnen <i>et al.</i>, 2006; O’Flaherty <i>et al.</i>, 2016)</a>. This is used to remove among others <i>E. coli</i>. Since we do not want any chemicals to potentially interfere with our biolenses, we have chosen not to use any of these chemicals and to test whether autoclaving or UV sterilization would be a good option.</p>
+                                            <p>To sterilize, different options are available. For example alcohols, chlorine and chlorine compounds are used within healthcare and autoclaving is a much-used method in laboratory settings. Besides that, UV sterilization is a much-used method in water treatment <a href="#references">(Chang <i>et al.</i>, 1985; Hijnen <i>et al.</i>, 2006; O’Flaherty <i>et al.</i>, 2016)</a>. This is used to remove among others <i>E. coli</i>. Since we do not want any chemicals to potentially interfere with our biolenses, we have chosen not to use any of these chemicals and to test whether autoclaving or UV sterilization would be a good option.</p>
-<h3>Methods</h3>
+                                            <h3>Methods</h3>
-<h4>Autoclaving biolenses</h4>
+                                            <h4>Autoclaving biolenses</h4>
-<p>Aliquots of cells were autoclaved before imaged with scanning electron microscopy (SEM). </p>
+                                            <p>Aliquots of cells were autoclaved before imaged with scanning electron microscopy (SEM). </p>
-<h4>UV sterilization</h4>
+                                            <h4>UV sterilization</h4>
-<p>A volume of 30 µL <i>E. coli</i> BL21 cells that were transformed with BolA in pBbA5c was plated on LB plates complemented with chloramphenicol. As controls for sterilization aliquots of 500 µL of the same cells were either heated at 95 °C for 10 minutes or washed with 500 µL 70% ethanol for three times. After sterilization, also 30 µL of these cells were plated on LB+Cm plates. Besides that, 30 µL of non sterilized cells were plated as well.</p>
+                                            <p>A volume of 30 µL <i>E. coli</i> BL21 cells that were transformed with BolA in pBbA5c was plated on LB plates complemented with chloramphenicol. As controls for sterilization aliquots of 500 µL of the same cells were either heated at 95 °C for 10 minutes or washed with 500 µL 70% ethanol for three times. After sterilization, also 30 µL of these cells were plated on LB+Cm plates. Besides that, 30 µL of non sterilized cells were plated as well.</p>
-<p>Two of the plates containing BolA transformed cells were UV sterilized in a Stratagene UV Stratalinker 1800. The manufacturer recommended power settings of 120000 µJ. In our experiment we have used a power of 120000 µJ and 240000µJ to sterilize our cells. </p>
+                                            <p>Two of the plates containing BolA transformed cells were UV sterilized in a Stratagene UV Stratalinker 1800. The manufacturer recommended power settings of 120000 µJ. In our experiment we have used a power of 120000 µJ and 240000µJ to sterilize our cells. </p>
-<p>All plates were incubated overnight at 37 °C.</p>
+                                            <p>All plates were incubated overnight at 37 °C.</p>
-<h3>Results</h3>
+                                            <h3>Results</h3>
-<h4>Autoclaving</h4>
+                                            <h4>Autoclaving</h4>
-<p>Cells containing the Sil_Sdom gene were autoclaved and subsequently imaged with SEM. </p>
+                                            <p>Cells containing the Sil_Sdom gene were autoclaved and subsequently imaged with SEM. </p>
-<center><figure>
+                                            <center><figure>
-    <img src="https://static.igem.org/mediawiki/2016/5/5b/T--TU_Delft--OmpA_autoclaved_with_silicic_acid.png" alt="">
+                                                    <img src="https://static.igem.org/mediawiki/2016/5/5b/T--TU_Delft--OmpA_autoclaved_with_silicic_acid.png" alt="">
-<figcaption>Figure 1. <i>E. coli</i> BL21 cells transformed with Sil_Sdom, provided with silicic acid and were autoclaved and subsequently imaged by with SEM. </figcaption>
+                                                    <figcaption>Figure 1. <i>E. coli</i> BL21 cells transformed with Sil_Sdom, provided with silicic acid and were autoclaved and subsequently imaged by with SEM. </figcaption>
-</figure></center>
+                                                </figure></center>
-<p>Since the SEM showed the cells were collapsed, and in no way could function as biolenses, another sterilization method was used. </p>
+                                            <p>Since the SEM showed the cells were collapsed, and in no way could function as biolenses, another sterilization method was used. </p>
-<h4>UV sterilization</h4>
+                                            <h4>UV sterilization</h4>
-<p>To test the effectivity of UV sterilization on our <i>E. coli</i> cells, a growth study after UV sterilization was performed. For this 30 µL BL21 cells transformed with BolA in pBbA5c was plated on LB plates complemented with chloramphenicol. These plates were UV sterilized in a Strategene UV Stratalinker 1800. As positive controls the same cells were heated for 10 minutes at 95 ºC or washed three times with 500 µL ethanol. As negative control the same cells were plated on a selective plate. </p>
+                                            <p>To test the effectivity of UV sterilization on our <i>E. coli</i> cells, a growth study after UV sterilization was performed. For this 30 µL BL21 cells transformed with BolA in pBbA5c was plated on LB plates complemented with chloramphenicol. These plates were UV sterilized in a Strategene UV Stratalinker 1800. As positive controls the same cells were heated for 10 minutes at 95 ºC or washed three times with 500 µL ethanol. As negative control the same cells were plated on a selective plate. </p>
-<p>None of the sterilized plates showed any growth, while the non sterilized plate did yield colonies.</p>
+                                            <p>None of the sterilized plates showed any growth, while the non sterilized plate did yield colonies.</p>
-<p>Besides the growth study, the cells were also imaged with SEM, which can be found in figure 2.
+                                            <p>Besides the growth study, the cells were also imaged with SEM, which can be found in figure 2.
-<center><figure>
+                                            <center><figure>
-<img src="https://static.igem.org/mediawiki/2016/3/30/T--TU_Delft--OmpA_UV_with_silicic_acid.png" alt="">
+                                                    <img src="https://static.igem.org/mediawiki/2016/3/30/T--TU_Delft--OmpA_UV_with_silicic_acid.png" alt="">
-<figcaption>Figure 2. <i>E. coli</i> BL21 cells transformed with Sil_Sdom, provided with silicic acid and were UV sterilized and subsequently imaged by with SEM. </figcaption>
+                                                    <figcaption>Figure 2. <i>E. coli</i> BL21 cells transformed with Sil_Sdom, provided with silicic acid and were UV sterilized and subsequently imaged by with SEM. </figcaption>
-    </figure></center>
+                                                </figure></center>
-<p>As shown in figure 2, the with silicate capturing of the cells remain undamaged after UV sterilization and could be used as biolenses now. </p>
+                                            <p>As shown in figure 2, the with silicate capturing of the cells remain undamaged after UV sterilization and could be used as biolenses now. </p>
-<h4>Discussion</h4>
+                                            <h4>Discussion</h4>
-<p>In this experiment, different methods to sterilize our biolenses were tested, as we wanted to be able to put our biological microlenses in real-world conditions. For this, we have both autoclaved and UV sterilized our cells. From the growth study, it can be concluded that the biolenses were successfully sterilized by UV sterilization. Besides that, the shape of the biolenses was not affected by UV sterilization as opposed to autoclaving. </p>
+                                            <p>In this experiment, different methods to sterilize our biolenses were tested, as we wanted to be able to put our biological microlenses in real-world conditions. For this, we have both autoclaved and UV sterilized our cells. From the growth study, it can be concluded that the biolenses were successfully sterilized by UV sterilization. Besides that, the shape of the biolenses was not affected by UV sterilization as opposed to autoclaving. </p>
-                                    <a href="#Biolenses" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#Biolenses').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
+                                            <a href="#Biolenses" class="btn btn-info" role="button" onclick="$('html,body').animate({scrollTop: $('#Biolenses').offset().top}, 'slow');" style="text-decoration:none; color:#f3f4f4; float:right;">Back to Top</a>
-                                </div>
+                                            </div>
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+                                            </div>
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-                        <div class="panel panel-default">
+                                            <div class="panel panel-default">
-                            <a class="collapsed" data-toggle="collapse" data-parent="#accordion" href="#collapseDrie" aria-expanded="false" aria-controls="collapseDrie">
+                                                <a class="collapsed" data-toggle="collapse" data-parent="#accordion" href="#collapseDrie" aria-expanded="false" aria-controls="collapseDrie">
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+                                                    <div class="panel-heading" role="tab" id="headingDrie">
-                                    <h4 class="panel-title">
+                                                        <h4 class="panel-title">
-                                        Improving solar cells with biolenses
+                                                            Improving solar cells with biolenses
-                                    </h4>
+                                                        </h4>
-                                </div>
+                                                    </div>
-                            </a>
+                                                </a>
-                            <div id="collapseDrie" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingDrie">
+                                                <div id="collapseDrie" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingDrie">
-                                <div class="panel-body">
+                                                    <div class="panel-body">
-                                    <h3>Introduction</h3>
+                                                        <h3>Introduction</h3>
-<h4>Ways to measure the efficiency of photovoltaics</h4>
+                                                        <h4>Ways to measure the efficiency of photovoltaics</h4>
-<p>There are different ways to measure the output of photovoltaic (PV) cells with accuracy and repeatability. The most used characteristic of PVs is their efficiency (Power Conversion Efficiency or PCE). It is defined as the energy produced from the device by the intended radiation from a light source. In order to measure the efficiency and produce acceptable results; a set protocols were set in the scientific community.</p>
+                                                        <p>There are different ways to measure the output of photovoltaic (PV) cells with accuracy and repeatability. The most used characteristic of PVs is their efficiency (Power Conversion Efficiency or PCE). It is defined as the energy produced from the device by the intended radiation from a light source. In order to measure the efficiency and produce acceptable results; a set protocols were set in the scientific community.</p>
-<p>Those protocols are relevant for the intended radiation and the measurements taken. For the intended radiation we make the assumptions of air mass 1.5 (dictates how the sun’s radiation is influenced by the atmosphere), the measured surface is 37 degrees from the equator and 48.2 degrees from Zenith. Finally, the radiation is 1000Wm<sup>-2</sup> <a href="#references">(Snaith, 2012) </a>.</p>
+                                                        <p>Those protocols are relevant for the intended radiation and the measurements taken. For the intended radiation we make the assumptions of air mass 1.5 (dictates how the sun’s radiation is influenced by the atmosphere), the measured surface is 37 degrees from the equator and 48.2 degrees from Zenith. Finally, the radiation is 1000Wm<sup>-2</sup> <a href="#references">(Snaith, 2012) </a>.</p>
-<p>The parameters that influence the PCE measurements can be found in characteristic J-V diagrams, plotting current density (A/cm2) against voltage (V) (figure 1). The graph contains the three basic parameters of PCE measurements. Those are the short circuit current (I<sub>sc</sub>), the open circuit voltage (V<sub>oc</sub>) and the Fill Factor (FF). This graph is obtained by scanning the photovoltaic’s current output within a range of voltages, usually from -1 Volts to +1 Volts. The V<sub>oc</sub> is the voltage when the current is zero and the I<sub>sc</sub> is the current when the voltage is zero. The Fill Factor represents how much power the solar cell is producing or its theoretical maximum. The theoretical maximum power for each solar cell is \( P_{ideal} = V_{oc} \times I_{sc} \) and the maximum power is \( P_{max} = V_{max} \times I_{max}\) then the Fill Factor is defined as \(FF = \frac{P_{max}}{P_{ideal}} \). The Fill Factor is a defining term of the behavior of the solar cells <a href="#references"> (Grossiord, Kroon, Andriessen, & Blom, 2012)</a>.</p>
+                                                        <p>The parameters that influence the PCE measurements can be found in characteristic J-V diagrams, plotting current density (A/cm2) against voltage (V) (figure 1). The graph contains the three basic parameters of PCE measurements. Those are the short circuit current (I<sub>sc</sub>), the open circuit voltage (V<sub>oc</sub>) and the Fill Factor (FF). This graph is obtained by scanning the photovoltaic’s current output within a range of voltages, usually from -1 Volts to +1 Volts. The V<sub>oc</sub> is the voltage when the current is zero and the I<sub>sc</sub> is the current when the voltage is zero. The Fill Factor represents how much power the solar cell is producing or its theoretical maximum. The theoretical maximum power for each solar cell is \( P_{ideal} = V_{oc} \times I_{sc} \) and the maximum power is \( P_{max} = V_{max} \times I_{max}\) then the Fill Factor is defined as \(FF = \frac{P_{max}}{P_{ideal}} \). The Fill Factor is a defining term of the behavior of the solar cells <a href="#references"> (Grossiord, Kroon, Andriessen, & Blom, 2012)</a>.</p>
-<center><figure>
+                                                        <center><figure>
-<img src="https://static.igem.org/mediawiki/2016/6/66/T--TU_Delft--Fill_Factor.png" alt="Fill Factor of PV devices">
+                                                                <img src="https://static.igem.org/mediawiki/2016/6/66/T--TU_Delft--Fill_Factor.png" alt="Fill Factor of PV devices">
-<figcaption>Figure 1. Characteristic curve of PV measurements. </figcaption>
+                                                                <figcaption>Figure 1. Characteristic curve of PV measurements. </figcaption>
-    </figure></center>
+                                                            </figure></center>
-<h4>Equipment and proposed measurements</h4>
+                                                        <h4>Equipment and proposed measurements</h4>
-<p>In order to achieve those reproducible and accurate measurement of efficiency a certain type of equipment complemented with previously mentioned protocols need to be used. The equipment used to simulate the aforementioned conditions is called <strong>Solar Simulator</strong>, which is basically a big Xenon lamp with well-defined light power output. In order to calibrate the simulator a ‘standard’ cell is used with well-defined properties to measure the exact output of the simulator in each measurement. Finally, the tested device must be placed perpendicular to the light with nothing restricting the light path and not moved during the whole testing. Using this set of equipment, we are sure that we conduct all the experiments under the same scientifically accepted conditions so we remove a factor of uncertainty to our experimental results.</p>
+                                                        <p>In order to achieve those reproducible and accurate measurement of efficiency a certain type of equipment complemented with previously mentioned protocols need to be used. The equipment used to simulate the aforementioned conditions is called <strong>Solar Simulator</strong>, which is basically a big Xenon lamp with well-defined light power output. In order to calibrate the simulator a ‘standard’ cell is used with well-defined properties to measure the exact output of the simulator in each measurement. Finally, the tested device must be placed perpendicular to the light with nothing restricting the light path and not moved during the whole testing. Using this set of equipment, we are sure that we conduct all the experiments under the same scientifically accepted conditions so we remove a factor of uncertainty to our experimental results.</p>
-<h3>Methods</h3>
+                                                        <h3>Methods</h3>
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-                    <!-- -Short introduction on our experiments and methods here -->
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-        <span class="anchor" id="references"></span>
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+                                            <div class="references container">
-            <h4 class="footer-title">References</h4>
+                                                <h4 class="footer-title">References</h4>
-            <ol>
+                                                <ol>
-                <li>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. </li>
+                                                    <li>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. </li>
-                <li>Cha, J. N., Shimizu, K., Zhou, Y., Christiansen, S. C., Chmelka, B. F., Stucky, G. D., & Morse, D. E. (1999). Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proceedings of the National Academy of Sciences, 96(2), 361-365. </li>
+                                                    <li>Cha, J. N., Shimizu, K., Zhou, Y., Christiansen, S. C., Chmelka, B. F., Stucky, G. D., & Morse, D. E. (1999). Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proceedings of the National Academy of Sciences, 96(2), 361-365. </li>
-                <li>Chang, J. C., Ossoff, S. F., Lobe, D. C., Dorfman, M. H., Dumais, C. M., Qualls, R. G., & Johnson, J. D. (1985). UV inactivation of pathogenic and indicator microorganisms. <i>Applied and environmental microbiology, 49</i>(6), 1361-1365.</li>
+                                                    <li>Chang, J. C., Ossoff, S. F., Lobe, D. C., Dorfman, M. H., Dumais, C. M., Qualls, R. G., & Johnson, J. D. (1985). UV inactivation of pathogenic and indicator microorganisms. <i>Applied and environmental microbiology, 49</i>(6), 1361-1365.</li>
-                <li>Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.</li>
+                                                    <li>Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33-38.</li>
-                <li>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-a Journal of the American Chemical Society, 127(45), 15749–15755.</li>
+                                                    <li>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-a Journal of the American Chemical Society, 127(45), 15749–15755.</li>
-                <li>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. </li>
+                                                    <li>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. </li>
-                <li>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. </li>
+                                                    <li>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. </li>
-                <li>Electron microscopy. (2013). Slideshare.net. Retrieved 15 October 2016, from http://www.slideshare.net/suniu/electron-microscopy</li>
+                                                    <li>Electron microscopy. (2013). Slideshare.net. Retrieved 15 October 2016, from http://www.slideshare.net/suniu/electron-microscopy</li>
-                <li>Fan, X., & Yun, S.-H. (2014). The potential of optofluidic biolasers. Nature methods, 11(2), 141-147. </li>
+                                                    <li>Fan, X., & Yun, S.-H. (2014). The potential of optofluidic biolasers. Nature methods, 11(2), 141-147. </li>
-                <li>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.</li>
+                                                    <li>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.</li>
-                <li>Friel, J. J., & Lyman, C. E. (2006). Tutorial review: X-ray mapping in electron-beam instruments. Microscopy and Microanalysis, 12(01), 2-25.</li>
+                                                    <li>Friel, J. J., & Lyman, C. E. (2006). Tutorial review: X-ray mapping in electron-beam instruments. Microscopy and Microanalysis, 12(01), 2-25.</li>
-                <li>Gather, M. C., & Yun, S. H. (2011). Single-cell biological lasers. Nature Photonics, 5(7), 406-410. </li>
+                                                    <li>Gather, M. C., & Yun, S. H. (2011). Single-cell biological lasers. Nature Photonics, 5(7), 406-410. </li>
-                <li>Grossiord, N., Kroon, J. M., Andriessen, R., & Blom, P. W. M. (2012). Degradation mechanisms in organic photovoltaic devices. <i>Organic Electronics, 13</i>(3), 432–456. http://doi.org/10.1016/j.orgel.2011.11.027</li>
+                                                    <li>Grossiord, N., Kroon, J. M., Andriessen, R., & Blom, P. W. M. (2012). Degradation mechanisms in organic photovoltaic devices. <i>Organic Electronics, 13</i>(3), 432–456. http://doi.org/10.1016/j.orgel.2011.11.027</li>
-                <li>Hardy, J., & Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297(5580), 353-356. </li>
+                                                    <li>Hardy, J., & Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297(5580), 353-356. </li>
-                <li>Heintzmann, R., & Ficz, G. (2006). Breaking the resolution limit in light microscopy. Briefings in functional genomics & proteomics, 5(4), 289-301. </li>
+                                                    <li>Heintzmann, R., & Ficz, G. (2006). Breaking the resolution limit in light microscopy. Briefings in functional genomics & proteomics, 5(4), 289-301. </li>
-                <li>Hijnen, W. A. M., Beerendonk, E. F., & Medema, G. J. (2006). Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo) cysts in water: a review. <i>Water research</i>, 40(1), 3-22.</li>
+                                                    <li>Hijnen, W. A. M., Beerendonk, E. F., & Medema, G. J. (2006). Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo) cysts in water: a review. <i>Water research</i>, 40(1), 3-22.</li>
-                <li>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. </li>
+                                                    <li>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. </li>
-                <li>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. </li>
+                                                    <li>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. </li>
-                <li>Jutteau, S., Paire, M., Proise, F., Lombez, L., & Guillemoles, J. F. (2015, 14-19 June 2015). Micro solar concentrators: Design and fabrication for microcells arrays. Paper presented at the Photovoltaic Specialist Conference (PVSC), 2015 IEEE 42nd</li>
+                                                    <li>Jutteau, S., Paire, M., Proise, F., Lombez, L., & Guillemoles, J. F. (2015, 14-19 June 2015). Micro solar concentrators: Design and fabrication for microcells arrays. Paper presented at the Photovoltaic Specialist Conference (PVSC), 2015 IEEE 42nd</li>
-                <li>Kim, E.-J., & Yoo, S.-K. (1998). Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein. Biotechnology techniques, 12(3), 197-201.</li>
+                                                    <li>Kim, E.-J., & Yoo, S.-K. (1998). Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein. Biotechnology techniques, 12(3), 197-201.</li>
-<li>Kwakernaak, I. C., & Sloof, I. W. G. (2005). Introduction in the use and application of Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy in materials., (November), 1–14.</li>
+                                                    <li>Kwakernaak, I. C., & Sloof, I. W. G. (2005). Introduction in the use and application of Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy in materials., (November), 1–14.</li>
-<li>Lakowicz, J. R. (2013). Principles of fluorescence spectroscopy: Springer Science & Business Media.</li>
+                                                    <li>Lakowicz, J. R. (2013). Principles of fluorescence spectroscopy: Springer Science & Business Media.</li>
-                <li>Li, C.-W., Chu, S., & Lee, M. (1989). Characterizing the silica deposition vesicle of diatoms. Protoplasma, 151(2-3), 158-163. </li>
+                                                    <li>Li, C.-W., Chu, S., & Lee, M. (1989). Characterizing the silica deposition vesicle of diatoms. Protoplasma, 151(2-3), 158-163. </li>
-                <li>McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., & Burgess, A. (2014). Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events. Cell Cycle, 13(9), 1400-1412. </li>
+                                                    <li>McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., & Burgess, A. (2014). Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events. Cell Cycle, 13(9), 1400-1412. </li>
-                <li>Müller, W. E. G. (2003). Silicon biomineralization.</li>
+                                                    <li>Müller, W. E. G. (2003). Silicon biomineralization.</li>
-                <li>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.</li>
+                                                    <li>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.</li>
-                <li>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. </li>
+                                                    <li>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. </li>
-                <li>Müller, W. E. (2011). Molecular biomineralization: aquatic organisms forming extraordinary materials: Springer Science & Business Media.</li>
+                                                    <li>Müller, W. E. (2011). Molecular biomineralization: aquatic organisms forming extraordinary materials: Springer Science & Business Media.</li>
-                <li>Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).</li>
+                                                    <li>Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).</li>
-                <li>Nam, M., Kim, K., Lee, J., Yang, S. S., & Lee, K.-K. (2013). Solar cell packaged by a microlens array and its long-term optical efficiency enhancement. Paper presented at the Proc. of SPIE Vol.</li>
+                                                    <li>Nam, M., Kim, K., Lee, J., Yang, S. S., & Lee, K.-K. (2013). Solar cell packaged by a microlens array and its long-term optical efficiency enhancement. Paper presented at the Proc. of SPIE Vol.</li>
-                <li>O’Flaherty, E., Membré, J. M., & Cummins, E. (2016). META-ANALYSIS OF THE EFFECT OF UV WATER TREATMENT ON ESCHERICHIA COLI. <i>BIOSYSTEMS AND FOOD ENGINEERING RESEARCH REVIEW 21</i>, 147.</li>
+                                                    <li>O’Flaherty, E., Membré, J. M., & Cummins, E. (2016). META-ANALYSIS OF THE EFFECT OF UV WATER TREATMENT ON ESCHERICHIA COLI. <i>BIOSYSTEMS AND FOOD ENGINEERING RESEARCH REVIEW 21</i>, 147.</li>
-                <li>Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).</li>
+                                                    <li>Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).</li>
-                <li>Nellist, P. D. (2011). The principles of STEM imaging. In Scanning Transmission Electron Microscopy (pp. 91-115). Springer New York.</li>
+                                                    <li>Nellist, P. D. (2011). The principles of STEM imaging. In Scanning Transmission Electron Microscopy (pp. 91-115). Springer New York.</li>
-                <li>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. </li>
+                                                    <li>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. </li>
-                <li>Shcherbo, D., Merzlyak, E. M., Chepurnykh, T. V, Fradkov, A. F., Ermakova, G. V, Solovieva, E. a, … Chudakov, D. M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nature Methods, 4(9), 741–746. http://doi.org/10.1038/nmeth1083</li>
+                                                    <li>Shcherbo, D., Merzlyak, E. M., Chepurnykh, T. V, Fradkov, A. F., Ermakova, G. V, Solovieva, E. a, … Chudakov, D. M. (2007). Bright far-red fluorescent protein for whole-body imaging. Nature Methods, 4(9), 741–746. http://doi.org/10.1038/nmeth1083</li>
-                <li>Snaith, H. J. (2012). The perils of solar cell efficiency measurements, <i>6</i>(June), 337–340.</li>
+                                                    <li>Snaith, H. J. (2012). The perils of solar cell efficiency measurements, <i>6</i>(June), 337–340.</li>
-                <li>Svelto, O., & Hanna, D. C. (1976). Principles of lasers: Springer.</li>
+                                                    <li>Svelto, O., & Hanna, D. C. (1976). Principles of lasers: Springer.</li>
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Difference between revisions of "Team:TU Delft/Project"

Revision as of 10:06, 17 October 2016

Opticoli

The new age of optics: Producing biological lenses and lasers to improve microscopy

Project Description

Limitations of microscopy

Improving microscopy using biology

Synthetic biology to produce biological lasers

Synthetic biology to produce biological lenses

Experiments and results

Expression of different fluorophores

Introduction

Experiments & Results

Emission spectra of different fluorophores

Introduction

Methods

Results and Discussion

The effect of expression of fluorophores on growth

Introduction

Methods

Results and Discussion

Discussion & Conclusions

Coating the cell in polysilica using silicatein

Introduction

Experiments & Results

Rhodamine staining of silica covered cells

Introduction

Methods

Results and discussion

Imaging of silicatein-expressing cells using TEM

Introduction

Methods

Results and discussion

Imaging of silicatein-expressing cells using SEM

Introduction

Methods

Results and Discussion

Analysis of physical properties of silica covered cells using AFM

Introduction

Methods

Results and discussion

Viability experiments of silica covered cells

Introduction

Methods

Results and Discussion

Discussion & Conclusions

Engineering a biological laser

Introduction

Experiments & Results

Imaging our biolaser using our home-built setup

Introduction

Methods

Results & discussion

Intensity measurements of laser cells

Introduction

Methods

Results and discussion

Discussion & Conclusions

Engineering biological lenses

Introduction

Experiments & Results

Influencing cell shape for round lenses

Introduction & background

Methods

Results and discussion

Sterilization of the biolenses

Introduction

Methods

Autoclaving biolenses

UV sterilization

Results

Autoclaving

UV sterilization

Discussion

Improving solar cells with biolenses

Introduction

Ways to measure the efficiency of photovoltaics

Equipment and proposed measurements

Methods

Results