BioBuilding: Synthetic Biology for Students: Lab 3: Difference between revisions
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==Part I: TinkerCell== | ==Part I: TinkerCell== | ||
Computer-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 | Computer-aided design (CAD) 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 to see how they perform. Biological engineers have fewer good CAD tools at their disposal. More often, they must 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 computer first? And then, with some good candidate designs in hand, we could turn to the bench with more confidence, having eliminated the clear failures. | ||
[[Image:TInkerCell.png|thumb|left| [http://www.tinkercell.com | [[Image:TInkerCell.png|thumb|left| [http://www.tinkercell.com TinkerCell] ]] | ||
One early effort at a CAD tool for synthetic biology is [http://www.tinkercell.com | One early effort at a CAD tool for synthetic biology is [http://www.tinkercell.com TinkerCell,] developed by engineers at the University of Washington. TinkerCell allows you to visually construct and then simulate/analyze a biological network. Using the following instructions, you can use TinkerCell to build the bacterial photography system (or at least a simplified model of it). For those who would like to read more about the TinkerCell CAD tool, you can find the details in [http://www.jbioleng.org/content/3/1/19 this article] from the Journal of Biological Engineering. | ||
=== | ===Getting Started with TinkerCell=== | ||
TinkerCell can be downloaded for free from [http://www.tinkercell.com/downloads-2#TOC-Download-current-versions this page.] Make sure you download the "current" version, not the "stable" version. The instructions in this tutorial were written for the Mac-based version of the program. If you are running TinkerCell on Windows or Linux, you may see some subtle differences. | |||
After you open the TinkerCell application, | After you open the TinkerCell application, begin familiarizing yourself with the basic operation of the program. In particular, try to use | ||
* the '''Molecules''' and ''' | * the '''Molecules''' and '''Reaction''' tabs: try to select 2 molecules from the molecules that are available. For example, click the "Enzyme" on the icon strip and then click the network canvas to place an enzyme. Repeat with a second molecule, selecting "Transcription Factor" from the icon strip and placing it on the network canvas. Next, choose the Reaction tab and select either activation or repression. Click on the enzyme first, then the transcription factor. If you chose activation, you'll be asked to choose between two mechanisms. A reaction arrow should appear. Next, if you like, try stamping out two receptor molecules and connect them with a different kind of regulation, or try making an enzyme catalyze a reaction with one or more small molecules. | ||
* the '''Parts''' and ''' | * the '''Parts''' and '''Regulation''' tabs: Choose the Parts tab and try stamping out a gene expression cassette, i.e. an operator (activator or repressor binding site), a promoter, an RBS, a protein coding sequence, and a terminator (optional). The parts do not need to be aligned when you place them on the network canvas, but if you drag them next to one another they should connect. You can then choose "Transcription Regulation" from the Regulation tab, and link the transcription factor you placed earlier to the gene's operator. A reaction arrow should appear. You can move the icons on the canvas, reshape the reaction arrows, and relabel the parts to your liking. Try using "Protein Production" from the Reaction tab to make the coding region of the gene produce a protein. | ||
===Instructions to build the bacterial photography system=== | |||
Now that you have the basic mechanics in hand, you can build the bacterial photography system. Follow the steps below, or use the additional tutorial linked [[Media:TinkerCellTutorial.pdf|here.]] ''Do not actually use the additional tutorial, it is not updated'' | |||
*'''Start this project''' on a new canvas. Select "New Canvas" from the File Menu, or click the new page icon on the top toolbar. | |||
*'''Assemble the reporter gene''': From the "Parts" tab, place an "Activator Binding Site", "Promoter," "RBS," and "Coding" icon on your canvas. Drag the parts next to each other so they snap together. | |||
*'''Name the reporter gene elements''': Click on the name below each part to rename it. The promoter should be named "PompC." The RBS can be left as is. The coding sequence can be renamed "LacZ". | |||
*'''Add the transcription factor''': From the "Molecules" tab, select "Transcription Factor" and print one onto the canvas. It will represent the phosphorylated form of OmpR, so rename it "OmpRp". | |||
*'''Visual appeal''': Select the OmpRp protein, and then from the "Edit" menu choose "Add decorator." A pop-up screen (shown on the right here) will display a choice of icons. From the "Decorators" tab, select "phosphorylation". | |||
[[Image:Add decorator.png|thumb|250px|right|Adding visual appeal]] | |||
*'''Activate Transcription of PompC with OmpRp''': From the "Regulation" tab, choose "Transcriptional Activation" and then click on OmpRp and the activator binding site just before PompC. Choose “Transcription Activation” from the pop-up menu (shown here). | |||
*'''Add the Cph8 light receptor''': From the | [[Image:AddTxnReg.png|thumb|right|Regulating transcription]] | ||
*'''Regulate OmpRp with Cph8''': From the | |||
*'''Add a Chassis''': From the | *'''Add the Cph8 light receptor''': From the "Molecules" tab, choose "Receptor" and print one on the canvas. Rename it "Cph8". | ||
*'''Add light''': From the | *'''Regulate OmpRp with Cph8''': From the "Regulation" tab, choose "Allosteric Inhibition" and then click on Cph8 and OmpRp. You can reshape the regulatory arrows and move the elements around the canvas as needed for clarity. | ||
** ''(In reality, in its nonphosphorylated form, Cph8 inhibits the activity of OmpRp. Thus, "phosphorylation-dephosphorylation cycle might be a better representation of reality, but "allosteric inhibition" works for our purposes and is simpler.)'' | |||
*'''Add a Chassis''': From the "Compartments" tab, choose “Cell” and print one on the canvas. Move and resize the cell so it encloses the transcription factor and the reporter gene. Leave the Cph8 receptor in the cell membrane. | |||
*'''Add light''': From the "Molecules" tab, choose "Small Molecule" and print one on the canvas. Rename it "Light" and connect it to Cph8 with an activation arrow from the "Reaction" tab. | |||
===Whew!=== | ===Whew!=== |
Revision as of 00:58, 25 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 (CAD) 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 to see how they perform. Biological engineers have fewer good CAD tools at their disposal. More often, they must 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 computer first? And then, with some good candidate 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, developed by engineers at the University of Washington. TinkerCell allows you to visually construct and then simulate/analyze a biological network. Using the following instructions, you can use TinkerCell to build the bacterial photography system (or at least a simplified model of it). For those who would like to read more about the TinkerCell CAD tool, you can find the details in this article from the Journal of Biological Engineering. Getting Started with TinkerCellTinkerCell can be downloaded for free from this page. Make sure you download the "current" version, not the "stable" version. The instructions in this tutorial were written for the Mac-based version of the program. If you are running TinkerCell on Windows or Linux, you may see some subtle differences. After you open the TinkerCell application, begin familiarizing yourself with the basic operation of the program. In particular, try to use
Instructions to build the bacterial photography systemNow that you have the basic mechanics in hand, you can build the bacterial photography system. Follow the steps below, or use the additional tutorial linked here. Do not actually use the additional tutorial, it is not updated
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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