Transcript ¡¡JerzyIdeas!!

Sample of ideas for iGEM 2009
Jerzy Szablowski
May 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:
[email protected]
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 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. 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
Engineered yeast cells
Engineered yeast cells
Engineered yeast cells
Computer
An array of microelectrodes
Engineered yeast cell:
One electrode changes voltage
Calcium rushes into the
stimulated cell and causes
an indicator to fluoresce.
Voltage sensitive Indicator
Voltage sensitive ion channel
Ca2+ efflux pump
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.
Selected
calcium dye
kd
Change of fluorescence Brightness
Excitation/Emission
Calcium Green 1 190nm
increases 100-fold upon 5x brighter
binding Ca2+, no auto
than Fluo-3
fluorescence [1]
490nm/530nm
Fluo-3
390nm
Increases 100-fold upon 1x Fluo3
binding Ca2+
506nm/526nm
Fluo-4
345nm
Increase 100-Fold upon
binding Ca2+
494nm/516nm
2x Fluo-3
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>.
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+ ATPase
Sodium
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.
Equipment
Experimental
Final product
Fluorescent filter
Illumination for fluorescence readout
Electrode
Engineering
Yeast cells
Fluorescent
Microscope
CCFL lighting
(like in laptops)
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 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 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.