BioBuilding: Synthetic Biology for Students: Lab 3: Difference between revisions
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'''TINKERCELL TODO:''' | |||
* Optimize parameters to make graph look nice | |||
* Retake screenshots | |||
* Rewrite instructions for current modeling procedure to reflect changes in TinkerCell interface | |||
* Add extensions at end for further realism | |||
** LacZ protein production | |||
** Catalysis of reporter molecule | |||
** Degradation of reporter molecule | |||
** (Realistic phosphorylation/dephosphorylation of Cph8 and OmpR? too advanced? more appropriate for college) | |||
* In graph section, give qualitative overview of kinetics, no diffeqs | |||
* Discussion questions: Explain what's happening in the graph, why each line has the shape it does. What happens if you change this slider and why? | |||
** Talk about gain? | |||
** On teacher side, link to a good kinetics primer with diffeqs in it | |||
* Prepare .tic files with simulation fully built and make available for download (where?) | |||
* Ask Deepak why do dependent variables appear in the slider box (or what am I misunderstanding) | |||
* Send bugs to Deepak | |||
'''OTHER TODO:''' | |||
* Move circuit gain exercise to a separate page as it is optional and kind of long; talk less about gain in system design and building-the-circuit sections | |||
* Format page nicely | |||
=Lab 3: Picture this= | =Lab 3: Picture this= | ||
Explore an engineered biological system through a computer simulation, an electronics building kit, and a real-life example. | |||
[[Image:PictureThis.png]] | [[Image:PictureThis.png]] |
Revision as of 11:44, 15 July 2011
Eau That Smell Lab |
TINKERCELL TODO:
OTHER TODO:
Lab 3: Picture thisExplore an engineered biological system through a computer simulation, an electronics building kit, and a real-life example. Acknowledgments: This lab was developed with MIT's undergraduate lab subject 20.109, in collaboration with extraordinary biological engineers: Jeff Tabor, Deepak Chandran, Reshma Shetty, & Steve Wasserman ObjectivesBy the conclusion of this laboratory investigation, the student will be able to:
IntroductionPart I: TinkerCellComputer-aided design is a hallmark of several mature engineering disciplines, like mechanical engineering or civil engineering. These engineers can rely on computer simulations to reliably predict the behavior of a car or a bridge, rather than run a hundred cars into walls or over bridges to see how those cars and bridges do. Biological engineers have fewer good CAD tools at their disposal. More often, at least for now, run laboratory experiments to test a system. But wouldn't it be nice (and quicker and less expensive too!) to try a few things on a laptop first? And then, with some good biological designs in hand, we could turn to the bench with more confidence, having eliminated the clear failures. One early effort at a CAD tool for synthetic biology is Tinkercell. This program was developed by engineers at the University of Washington and it allows you to visually construct and then simulate/analyze a biological network. With the instructions that are here, you can use Tinkercell to build the genetic circuit that underlies the bacterial photography system. For those who would like to read more about the TinkerCell CAD tool, you can find the details in a Journal of Biological Engineering article that you can find here. Let's get startedThe TinkerCell application can be downloaded for free from here. The instructions in this tutorial were written for the Mac-based version of the program. If you are running TinkerCell on a PC, you may see some subtle differences. After you open the TinkerCell application, you should begin familiarizing yourself with the basic operation of the program. In particular, try to use
If you get stuck or in trouble, try the undo icon. You’ll find that useful function 8th from the right at the top of the program. Instructions to visually build the bacterial photography systemNow that you have some of the basic mechanics in hand, try to visually construct the bacterial photography system according to the tutorial linked here.
Whew!Now that you've dragged, dropped and connected all the parts in the bacterial photography system, it's time to consider what you've got. Did you learn more about the system by building it? Were there some approximations you made as you built it? Does it matter that there some parts left out for simplicity, for instance the phosphorylated form of Cph8? Can you see places where different parts might be interesting to try? How big is the gap between the things we know about, and the things we need to learn more about? Are there any experiments you'd like to try? What reactions could you measure? What could you mutate to improve? If you'd like, you can go on to use the TinkerCell program to model the answers to some of these questions.... Instructions to model the bacterial photography systemIf you would like to try to simulate the operation of the system, that can be done in Tinkercell. Find the green arrow from the icons at the top of the program (7th from the right) and choose "Deterministic" from the Simulation options. This selection should open a window with sliders and a second window with a graph. Take a few minutes to familiarize yourself with the color coding of the output graph. What are the axes? What color line can you expect for the level of Cph8, of OmpRp, etc? Take another few minutes to familiarize yourself with the sliders. What numbers are shown? What happens to the numbers if you move a slider for one of the reactions? What changes to the biology of the system are you making when you use the sliders? Now think about what the model is showing you and how you can use that information. Are there new experiments you'd like to try if you could? Are there experiments that don't seem worth doing? If there are impossible outputs from the simulation, then are there changes to the model itself you'd like to make? More regulatory reactions? Fewer? Putting it all togetherThe last question to ask yourself as you finish this modeling of the bacterial photography system is this: was it worth it? You've just spent an hour or two working with this program. In that time maybe you've learned about the system you're studying, decided on some future experiments, or designed some novel variations that, at least as modeled, seem worth pursuing. Part II. Electronic vs Biological CircuitsIn this activity, we'll explore signaling in the context of an electrical circuit. As you work through this exercise, consider how the lessons learned from experimenting with an electronic circuit would map to the engineering of biological systems. You will be given a kit to construct this circuit. SafetySafety for you: In this exercise, you'll be working with circuits connected to a battery. Although you are unlikely to seriously injure yourself, you should make a habit of unplugging / powering-off the circuit before you touch it. Safety for the circuit components:
System designThis system is a fairly simple one, consisting of only a few components. In contrast to the bacterial photography system in which the signal is propagated through protein activities, here signals are propagated as either voltage or current. When light hits the photodiode, it generates a current signal. The OpAmp takes in this current signal and produces a voltage, which signals the LED to produce light. As you can see in the schematic, the circuit contains the following parts:
Move part numbers to teachers notes only.
Intro to BreadboardsYou will be building this circuit on a breadboard, which is a much cleaner way to construct circuits than just wiring everything together. Start by building a very simple, "Hello World" circuit to understand intuitively how breadboarding works. (If you have used a breadboard before, feel free to skip this section.) Take a look at the rows of holes on your breadboard. In the middle, the holes are arranged in short rows of five. The five holes in each row are all connected to each other (via thin strips of metal inside the breadboard). On the sides are the rails, or long rows of holes marked with red or blue lines and a plus or minus sign. All the holes in a rail are connected to each other. Rails allow you to conveniently serve power/ground to many locations at once. For example:
Hooking up an LEDRemember that the LED only allows current to flow through it in one direction. If you try to put current through it backwards, it won't work. (But it won't damage the LED either, so feel free to try!) The resistor is needed to limit the current through the LED. Too much current can damage it. (The LEDs in this kit are strong enough that they won't burn out instantly if you connect them straight across the battery, but using a resistor is still recommended.) You could also put the resistor on the power side and the LED on the ground side; it makes no difference. First, deliver power from the battery to the rails on either side of the breadboard. Use the binding posts to connect the battery leads to wires, and plug the wires into the rails. Unscrew the plastic part of the binding post, insert a lead and a wire into the hole, and tighten the plastic part. (Get a good connection -- make sure you are clamping down on a metal wire and not on the plastic insulation around each wire.) Now plug in the LED and the resistor. Push in the wires firmly to make a good connection. You should see light. If not, check your connections, make sure your battery is not dead, etc. Let's start building
Examining the system behaviorResistance = 10 MΩRight now, the OpAmp's output and minus input are connected with a 10 MΩ resistor.
The range of flashlight intensities that can hold the LED 1/2 lit is a measure of the "gain" in the system--where a narrow range of fully on to fully off is "digital" (switch-like) behavior, while a wide range of flashlight intensities that hold the LED 1/2 on is more "analog" (dial-like) behavior. A circuit with very tight fully-on-or-fully-off behavior is more "digital", or switch-like, while a circuit where the LED can have a wide middle range of brightness is more "analog", or dial-like. The range of flashlight intensities that can hold the LED half-lit is a measure of the "gain" or strength of the amplifier. We can tune this gain by changing the value of the gain resistor. 4. Sketch a graph with flashlight intensity on the x-axis and LED light intensity on the y-axis. At infinite resistance in place, is the circuit's behavior better described as a switch or a dial? Resistance = 0ΩReplace the 10MΩ resistor with a wire.
Resistance = infinite ΩRemove the wire connecting the OpAmp's output to its negative input.
Discussion
Part III. U-do-it Bacterial PhotographDecide what image you would like to develop as a bacterial photograph. Remember that the goal is to have each cell growing distinctly in the light or dark. Light can bounce around edges and may blur the resulting image if the black and white are highly intermingled. In general, it’s better to have a dark background and a light image rather than the other way around. Once you have decided on an image, generate a computer file with this image and print it to a transparency. To darken the dark parts of your photo, you might want to print it on two transparencies and use them both to mask the Petri dish. The diameter of the petri dish is less than 3 inches across so your image must be smaller than this. Email info AT BioBuilder DOT org to say that a transparency is being sent, and in a few days, a jpg file with your bacterial photo, or the plate itself if that's possible, will be sent back to you.
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