Sample of ideas for iGEM 2009

1. Sample of ideas for iGEM 2009 JerzySzablowski May 2009

2. Short Introduction Hi, my name is JerzySzablowski, I was born in Poland, but I moved to the United States to study at MIT. I finished a couple days ago with a Bsc In Biological Engineering, concentrating mostly on protein engineering and biomaterials. In my free time, I enjoy photography, cross country running and squash. If you have any questions regarding the proposals, let me know at: jszab@mit.edu

3. Idea 1 Cell based computer display This is an idea which I personally like the most. I have bunch of other ones listed with short descriptions on the last page. Please let me know which ones, if any, sound much cooler than this one and I will write more detailed descriptions.

4. Yeast based computer monitor • What are the ways of getting an arbitrary spatial pattern of fluorescent cells? • Simple methods: • bacterial photographic system from Voigt/Ellington? • Use a smart method of diffusing the activators and use Weiss fluorescence systems? • Co-express ligands and fluorescent/luminescent molecules, nanopattern the surface? Levskaya, A., et al. (2005) Synthetic biology: engineering Escherichia coli to see light. Nature, 438, 441–442 Basu, S. , Gerchman, Y. , Collins, C. H. , Arnold, F. H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature434, 1130–1134 (2005) Asthagiri group, California Institute of Technology

5. What is wrong with all of these examples? • It will take minutes or even hours to refresh an image. • There is no electrical input that will allow you to control the image from an electronic component. • The next several slides will include the simplest (but not the best) approach to overcome these issues.

6. Yeast based monitor – general idea • Cellular signaling operates on a millisecond timescale = appropriate for a computer display. • It can be visualized using many of the fluorescent/luminescent techniques. Actuate a fast and simple signal (for example an electrical impulse) into cellular signaling that then can be visualized using one of the many fluorescent indicators.

7. Outline of the yeast based monitor Engineered yeast cells Engineered yeast cells Engineered yeast cells Computer An array of microelectrodes One electrode changes voltage Calcium rushes into the stimulated cell and causes an indicator to fluoresce. Engineered yeast cell: Voltage sensitive Indicator Voltage sensitive ion channel Ca2+ efflux pump

8. Calcium indicators - chemical Pros: work out of the box, simple to use. Robust and provide high signal/noise ratio. Many choices. Cons: not genetically encoded, need to be replenished after a while. More dyes described in: R. Madelaine Paredes, Julie C. Etzler, Lora Talley Watts, Wei Zheng and James D. Lechleiter Chemical calcium indicators Optical Methods in Calcium Signaling , Volume 46, Issue 3, November 2008, Pages 143-151 [1] <http://probes.invitrogen.com/handbook/sections/1903.html>.

9. Voltage sensitive calcium channels • There are quite a lot. At first we should use the Calcium channel from smooth, or cardiac muscles. Express: Ca2+ ATPase Sodium Calcium exchanger Express one of these channels in yeast: Then, we should see how much we can change the extracellular potential by the electrodes. We will need to ask neurobiologists. This kind of practice is done in deep brain stimulation, muscle activation etc.

10. Equipment Experimental Final product Fluorescent filter Illumination for fluorescence readout Electrode CCFL lighting (like in laptops) Engineering Yeast cells Fluorescent Microscope Array of electrodes connected to a computer

11. Alternatives? • Use of cardiac muscle = these respond to electrical stimulation by calcium influx. Thus, all we would need to engineer is an electrical stimulation array. • There are genetically encoded calcium indicators we could use (CaMeleon, Pericam…) but they are not as good as chemical ones. • We could use mechanical actuation: a small piezoelectric connected to an electrode. • We will need to tweak the signaling to avoid background calcium signaling (especially calcium waves) • Worst comes to worst, we can actuate chemicall and have a good local perfusion system or do this in a 1536 well plate. • We can base this system on bioluminescent proteins • We can also use another ion to avoid some of the problems. The last two points will require more protein engineering. I can explain what would be necessary if you would like me to.

12. Anticipated problems • The electrical potential will not be enough to depolarize the membrane and cause significant Ca2+ influx. • The electrical field will stimulate many cells around the electrode = low resolution display. • The response will be relatively slow depending on the system. • Baseline signaling in yeast will cause the display to be noisy.

13. Other ideas available upon request. • MRI gradient coil made of bacteria and bacteriophages. Allows for visualizing the presence of small magnetic particles that flow into the designated area. Everything is at micrometer scale. We still need a connection to a computer and an external RF coil. In short: Magnetotactic bacteria are patterned on a surface depending on their accumulation of magnetite core. The surface between them will be affected by a magnetic field. This field will cause a gradient if we nanopattern the surface properly, which can be used for the detection of the presence of bacteria expressing an MRI contrast agent such as Cytochrome P450-BM3. • Bacteria-based telegraph Unidirectional spread of an encoded signal – similar to the way neurons transduce information. I have bunch of ideas for this one. Why would it be useful? Because it could be used for in-vivo stimulation of a chosen site in the deep tissues of an animal model. Currently it is not possible. • Bacteriophage based scaffold for tissue engineering. Bacteriophages are evolvable construction units that can be functionalized with many useful materials, such as RGD peptides. They also can be evolve to change their mechanical properties or the way they act to differentiate stem cells. In short: I would like to crosslink the bacteriophages to each other and functionalize them with RGD peptides such that they can form macromolecular a scaffold for Tissue Engineering. Subsequently, they would be evolved in high throughput screen to influence physiology of cells in a fashion we would choose together. • Ca2+ gated bioluminescent switch. Marc Ostermeier made an allosterically controlled Beta-lactamase-Maltose Binding protein switch*. Upon introduction of maltose, the activity of Beta-lactamase drops down. The same can be done with a renillaluciferase – a protein that is capable of bioluminescing inside the living cells. I propose to fuse one of the termini of renillaluciferase to one of calmodulin. Upon elevation of calcium levels, calmodulin will change its conformation and thus the amount of coelenterazine that can enter into the enzymatic pocket. Careful rational design or directed evolution will allow us to maximize the contrast between off and on states. *Guntas, G. and M. Ostermeier. (2004) Creation of an allosteric enzyme by domain insertion. J. Mol Biol. 336:263-273.