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Revision as of 12:49, 21 September 2016

iGEM TU Delft

Producing biological lenses and lasers to improve microscopy

Microscopes have been around for hundreds of years and the technology behind these devices has been quickly developing over the past centuries. Microscopy has already helped us to image cells into great detail, which is essential for the identification of mechanisms behind diseases such as Alzheimer’s, of which we still don’t know the exact mechanism, but also for developing synthetic biology even further. In this age, the technology and knowledge of microscopy is no longer limiting for making detailed images of the cell; it’s the cells itself. When using fluorescence microscopy, a fluorescent cell only emits a limited number of photons, a part of this will not reach the detector. This year’s TU Delft team is using synthetic biology with the aim of improving fluorescence microscopy. There are two research lines: producing biological lenses and inventing a bacterial laser. Hover over the pictures underneath to find out more.

BIOLENSES

lenses

The goal of our biological microlenses is to increase the fraction of light captured by the detector of a microscope. Lenses are known to focus light onto a surface. By applying a layer of our biological microlenses on the detector of a microscope, we can increase the fraction of light captured. To produce microlenses, we expressed the enzyme silicatein in our cells, which catalyzes polymerization of silicic acid (Cha et al., 1999). This results in a biosilica layer around the cell (Muller et al., 2008), allowing the cell to function as a microlens. Since the shape of the lenses is a crucial property, we also overexpressed the gene bolA in our silica covered cells, which produces a round cell shape when overexpressed (Aldea & Concha, 1988), to produce round lenses. Apart from using the lenses for microscopy, we can also use the lenses for improving the efficiency of solar panels, thin lightweight cameras with high resolution or 3D screens.

BIOLASERS

laser

By turning a cell into a biolaser, we will increase the light intensity emitted by the fluorescent cell. The cell will then emit more photons without changing the fluorophore concentration. When more photons are emitted, more photons can be detected by the microscope. A laser works by resonating photons within a closed space, in this case a cell of E. coli. We approached this by expressing fluorescent proteins within our biosilica-covered cells we used for our biolenses. When exciting the fluorophores, a fraction of the photons are trapped inside the cell by the biosilica layer. When these photons meet other excited fluorescent proteins they cause them to emit a photon with the same wavelength and direction, this process is called ‘stimulated emission’ (Einstein, A. 1917) and results in light with a higher intensity and thus more emitted photons compared to conventional fluorescence.

  1. Aldea, M., & Concha, H. C. (1988). Identification, Cloning, and Expression of bolA, an ftsZ-Dependent Morphogene of Escherichia coli. Journal of Bacteriology.
  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. Biochemistry, 96, 361–365.
  3. Einstein, A. (1917): "Zur Quantentheorie der Strahlung". Physikalische Zeitschrift 18, 121-128
  4. Muller, W., Engel, S., Wang, X., Wolf, S., Tremel, W., Thakur, N., … Schrodel, H. (2008). Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-α gene. Biomaterials, 29(7), 771–779. http://doi.org/10.1016/j.biomaterials.2007.10.038

Parts

Below you will find all the parts listed that were created by the TU Delft team in the course of the iGEM 2015 competition.

Overview

This year IDT sponsored the synthesis of 20 kb for every iGEM team. For this reason, we did not clone our basic parts into the pSB1C3 plasmid backbone, but directly combined them in the synthesis of our composite parts. However, we experienced problems with 2 out of 20 gBlock genes during cloning which unfortunately in the end had to be dropped from the project.

Our initial plan to use GFP (BBa_I13522) to additionally color label the cells containing our plasmid DNA was dropped in favor of using RFP (I13521). In our experience the bacterial cells are intensively red when expressing this gene, which especially makes colony picking after transformation significantly more straightforward than with GFP. Based on this we want to encourage all coming iGEM teams to use RFP instead of GFP.

Since our project was about the induced formation of a biofilm from amyloid nanowires, most of our parts consist of the csgA gene or are variations of it. For instance, in order to mimick the characteristic adhesiveness of biofilms we added different affinity tags to CsgA.

TU Delft iGEM 2015 Parts

Basic & Composite parts

<groupparts>iGEM015 TU_Delft</groupparts>

Part Collection

CsgA multitool box

Biofilms are communities of bacteria that live together on a surface, surrounded by a protective layer, like dental plaque. Biofilms consist up to 85% of nanowires. These nanowires are composed of CsgA proteins. All the parts in this collection contain the CsgA gene controlled by a rhamnose inducible promoter. However, most have an additional function, like the His-tag for purification, or the hydroxyapatite tag, that gives the CsgA proteins better adhesive properties to teeth, thereby closer resembling dental plaque. Additionally, several biobricks contain RFP or GFP genes, to improve detection.

pRha + CsgA

pRha + CsgA & pTet+RFP

pRha + CsgA+His tag

pRha + CsgA+His tag & pTet+RFP

pRha + CsgA+Hydroxyapatite affinity tag

pRha + CsgA with Hydroxyapatite affinity tag & pTet RFP

pRha + fusion protein of CsgA with Mfp3 + His tag

pRha + fusion protein of CsgA with Mfp3 + His tag & pTET + RFP

pRha + fusion protein of Mfp5 with CsgA + His tag

pRha + fusion protein of Mfp5 with CsgA + His tag & pTET + RFP

pRha + CsgA & GFP in same operon