Sample of ideas for iGEM 2009

Jerzy SzablowskiMay 2009

Short Introduction

Hi, my name is Jerzy Szablowski, 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

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.

Yeast based computer monitorWhat 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. Nature 434, 1130–1134 (2005)

Asthagiri group, California Institute of Technology

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.

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.

Outline of the yeast based monitor

An array of microelectrodes

Engineered yeast cells

One electrode changes voltage

Engineered yeast cells

Calcium rushes into the stimulated cell and causes an indicator to fluoresce.

Engineered yeast cells

Engineered yeast cell:

Voltage sensitive Indicator

Voltage sensitive ion channel

Ca2+ efflux pump

Computer

Calcium indicators - chemical

Selected calcium dye

kd Change of fluorescence Brightness Excitation/Emission

Calcium Green 1 190nm increases 100-fold uponbinding Ca2+, no auto fluorescence [1]

5x brighter than Fluo-3

490nm/530nm

Fluo-3 390nm Increases 100-fold upon binding Ca2+

1x Fluo3 506nm/526nm

Fluo-4 345nm Increase 100-Fold upon binding Ca2+

2x Fluo-3 494nm/516nm

[1] <http://probes.invitrogen.com/handbook/sections/1903.html>.

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

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.

Voltage sensitive calcium channels• There are quite a lot. At first we should use the

Calcium channel from smooth, or cardiac muscles.

Express one of these channels in yeast:

Express:Ca2+ ATPaseSodium Calcium exchanger

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.

EquipmentExperimental

Electrode

Fluorescent Microscope

Illumination for fluorescence readout

Final product

CCFL lighting (like in laptops)

Fluorescent filter

EngineeringYeast cells

Array of electrodes connected to a computer

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.

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.

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 telegraphUnidirectional 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 renilla luciferase – a protein that is capable of bioluminescing inside the living cells. I propose to fuse one of the termini of renilla luciferase 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.