BioBuilding: Synthetic Biology for Students: Lab 3: Difference between revisions
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* Define and properly use molecular genetics terms: two component system, transcriptional activation, phosphorylation | * Define and properly use molecular genetics terms: two component system, transcriptional activation, phosphorylation | ||
* Relate the bacterial photography system to the two component signaling system. | * Relate the bacterial photography system to the two component signaling system. | ||
* Model a biological system using electronic parts and a computer program. | * Model a biological system using electronic parts and a computer program. | ||
* Explain the role that modeling can play in design, and name some ways that models differ from reality. | |||
==Introduction== | ==Introduction== | ||
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*'''Add the Cph8 light receptor''': From the "Molecules" tab, choose "Receptor" and place one on the canvas. Rename it "Cph8". | *'''Add the Cph8 light receptor''': From the "Molecules" tab, choose "Receptor" and place one on the canvas. Rename it "Cph8". | ||
*'''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. | *'''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. | ||
*'''Add the Beta-Gal protein''': From the "Molecules" tab, place an enzyme on the canvas and call it "BetaGal". Link it to the LacZ coding region using the "Protein Production" reaction. | *'''Add the Beta-Gal protein''': From the "Molecules" tab, place an enzyme on the canvas and call it "BetaGal". Link it to the LacZ coding region using the "Protein Production" reaction. | ||
*'''Add the colorful small molecule''': Place two more small molecules on the canvas. Connect them to your BetaGal protein using "Enzyme Catalysis" from the "Regulation" tab. Name the input molecule "SGal", and the output molecule "COLOR". Next, double-click on SGal to open a dialog box. Select the "Initial Conditions" tab and increase the concentration to 15. | *'''Add the colorful small molecule''': Place two more small molecules on the canvas. Connect them to your BetaGal protein using "Enzyme Catalysis" from the "Regulation" tab. Name the input molecule "SGal", and the output molecule "COLOR". Next, double-click on SGal to open a dialog box. Select the "Initial Conditions" tab and increase the concentration to 15. | ||
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Behind the shiny-looking front end of TinkerCell is some serious mathematical capability. We'll use the "Deterministic" simulator (i.e. we will be ignoring the random fluctuations that would occur in a real cell). Click the big green arrow in the top toolbar to run the system. | Behind the shiny-looking front end of TinkerCell is some serious mathematical capability. We'll use the "Deterministic" simulator (i.e. we will be ignoring the random fluctuations that would occur in a real cell). Click the big green arrow in the top toolbar to run the system. | ||
You should see a graph window and a second window with sliders. To clean up the graph by getting rid of some unnecessary lines, click on "Legend" in the graph window, and uncheck everything except Time, Light, Cph8, OmpRp, LacZ, BetaGal, and COLOR. ''(Everything else is either constant or an internal TinkerCell bookkeeping variable.)'' Take a few minutes to familiarize yourself with the output graph. | You should see a graph window and a second window with sliders. To clean up the graph by getting rid of some unnecessary lines, click on "Legend" in the graph window, and uncheck everything except Time, Light, Cph8, OmpRp, LacZ, BetaGal, and COLOR. ''(Everything else is either constant or an internal TinkerCell bookkeeping variable.)'' Take a few minutes to familiarize yourself with the output graph. You may want to answer discussion questions 1-3 at this step, instead of waiting until the end. | ||
====Tuning the system==== | ====Tuning the system==== | ||
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This 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: | This 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: | ||
#[[Media:Photodiode SpecSheet.pdf| Photodiode]] | #[[Media:Photodiode SpecSheet.pdf| Photodiode]]: a light sensor ''(analogous to the Cph8-OmpR signaling system)''. When light shines on the photodiode, its resistance decreases, and current flows through it. | ||
#[[Media:OpAmpDataSheet.pdf| OpAmp]] | #[[Media:OpAmpDataSheet.pdf| OpAmp]]: a signal propagator ''(analogous to the transcription/translation machinery that translates an OmpR signal into synthesis of LacZ)''. More generally, an OpAmp is a logic device that detects and amplifies a difference between the currents into its plus and minus inputs. With the addition of the feedback resistor connecting the output to the minus input, the OpAmp translates an incoming current signal into an outgoing voltage signal. | ||
#[http://www.breakup.de/resources/resistor.html Resistor]: component which resists current flow by producing a voltage drop across it. The voltage equals the current times the resistance of the resistor; thus, the resistance sets the "gain" or amplifier strength of the system. By varying the resistance, we can vary the circuit's sensitivity to light. ''(The resistor is analogous to the strength of the promoters and ribosome binding sites in the biological system, which raise or lower the efficiency of LacZ production.)'' | #[http://www.breakup.de/resources/resistor.html Resistor]: component which resists current flow by producing a voltage drop across it. The voltage equals the current times the resistance of the resistor; thus, the resistance sets the "gain" or amplifier strength of the system. By varying the resistance, we can vary the circuit's sensitivity to light. ''(The resistor is analogous to the strength of the promoters and ribosome binding sites in the biological system, which raise or lower the efficiency of LacZ production.)'' | ||
#[http://www.allelectronics.com/make-a-store/item/LED-2/GREEN-5MM-T1-3/4-LED//1.html LED]: a device with a detectable output ''(analogous to LacZ)''. A voltage drop across its terminals turns the green-colored light on. (The small 820Ω resistor is placed in line with the LED to ensure that the current through the LED isn't too high, which can fry the LED. This small resistor has no direct analogue in the bacterial photography system.) | #[http://www.allelectronics.com/make-a-store/item/LED-2/GREEN-5MM-T1-3/4-LED//1.html LED]: a device with a detectable output ''(analogous to LacZ)''. A voltage drop across its terminals turns the green-colored light on. (The small 820Ω resistor is placed in line with the LED to ensure that the current through the LED isn't too high, which can fry the LED. This small resistor has no direct analogue in the bacterial photography system.) | ||
<table border="1"> | <table border="1"> | ||
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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. | 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. | ||
=== | ===Building the bacterial photography circuit=== | ||
[[Image:Finishedcircuit.jpg|thumb|right|200px|'''Finished circuit.''']] | [[Image:Finishedcircuit.jpg|thumb|right|200px|'''Finished circuit.''']] |
Revision as of 07:26, 28 July 2011
Eau That Smell Lab |
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, & Kelly Drinkwater ObjectivesNEED TO REWRITE THESE / REWRITE DISCUSSION QUESTIONS TO MATCH -- in particular the first one By 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 we'll go on to simulate the photography system in action... Instructions to simulate the bacterial photography systemBehind the shiny-looking front end of TinkerCell is some serious mathematical capability. We'll use the "Deterministic" simulator (i.e. we will be ignoring the random fluctuations that would occur in a real cell). Click the big green arrow in the top toolbar to run the system. You should see a graph window and a second window with sliders. To clean up the graph by getting rid of some unnecessary lines, click on "Legend" in the graph window, and uncheck everything except Time, Light, Cph8, OmpRp, LacZ, BetaGal, and COLOR. (Everything else is either constant or an internal TinkerCell bookkeeping variable.) Take a few minutes to familiarize yourself with the output graph. You may want to answer discussion questions 1-3 at this step, instead of waiting until the end. Tuning the systemTurn to the slider window and start changing numbers, making notes of what effects you see. (If you screw something up, you can always close the graph/slider windows and re-run the simulation with default values.) Many of the variables in the slider window have straightforward names, like "light_step_height", but some are more opaque. In general, things named "Kd" are constants governing the strength of a regulatory interaction. "Kcat" is the catalysis power of the BetaGal enzyme.
Next, decide on some specific changes you would like to make to the graph, and then see if you can find the right combination of sliders to make those changes. For example:
Putting it all togetherYour teacher may ask you to answer some of the following questions.
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:
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. Building the bacterial photography circuit
Finally, if desired, continue to the Exploring Gain exercise. 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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