BioBuilding: Synthetic Biology for Students: Lab 3: Difference between revisions
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==Introduction== | ==Introduction== | ||
If you have not had a lesson on how the bacterial photography system works, go read the first half of the [[BioBuilding: Synthetic Biology for Students: Design Assignment | design assignment page]]. | |||
==Part I: TinkerCell== | ==Part I: TinkerCell== | ||
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====Tuning the system==== | ====Tuning the system==== | ||
Turn 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 | Turn 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 catalytic efficiency of the BetaGal enzyme. | ||
* | * Change the amount of SGal in the bacteria's environment (the "cel1_SGal" slider). What happens to the COLOR output? | ||
* Change the efficiency of the BetaGal enzyme (the "cel1_ec1_Kcat" slider). What happens to the output? | |||
* Decrease "Light_step_steepness", so that the light turns on gradually rather than all at once. Does this tell you more about how exactly the system responds when its input changes? | |||
* Try changing the light's step input to a sine wave input, and change the frequency. What happens to the output when the frequency is high? When the frequency is low? | * Try changing the light's step input to a sine wave input, and change the frequency. What happens to the output when the frequency is high? When the frequency is low? | ||
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# What are some approximations / simplifications that you made as you built the model? (For example, leaving out the phosphorylated form of Cph8.) Do these matter? | # What are some approximations / simplifications that you made as you built the model? (For example, leaving out the phosphorylated form of Cph8.) Do these matter? | ||
# What are the axes of the graph? What are the units of each quantity (concentration, rate, etc.)? (Check the listed units for each quantity in the summary menu on the right.) | # What are the axes of the graph? What are the units of each quantity (concentration, rate, etc.)? (Check the listed units for each quantity in the summary menu on the right.) | ||
# Consider each line on the graph. Does it go up, down, or stay static in the first half of the simulation (light off)? Does it change at the time the light turns on? Does it go up, down, or stay static in the second half of the simulation (light on)? | # Consider each line on the graph. Does it go up, down, or stay static in the first half of the simulation (light off)? Does it change at the time the light turns on? Does it go up, down, or stay static in the second half of the simulation (light on)? | ||
# Name four adjustments you made to the sliders, describe how they affected the shape of the lines on the graph, and why each adjustment made the difference that it did. | # Name four adjustments you made to the sliders, describe how they affected the shape of the lines on the graph, and why each adjustment made the difference that it did. | ||
## | ## Describe how you might carry out some of these adjustments in a real cell. | ||
# Why is it valuable for scientists to be able to model their experiments in this way? For engineers to be able to model their creations? Did you personally find it valuable? | |||
# What are some of the drawbacks of this modeling approach? Where does the analogy between a TinkerCell canvas and a biological system break down, so that the model no longer reflects reality? | # What are some of the drawbacks of this modeling approach? Where does the analogy between a TinkerCell canvas and a biological system break down, so that the model no longer reflects reality? | ||
# Did you encounter any bugs in TinkerCell? Are there features you would like to see? Describe each bug or feature | # Did you encounter any bugs in TinkerCell? Are there features you would like to see? Describe each bug or feature precisely. Collect all the bug reports / feature requests as a class, and [http://www.tinkercell.com/suggestions-bugs submit them to the TinkerCell development team.] | ||
# Finally, | # Finally, did this exercise suggest any modifications you would like to make to the system, or experiments you would like to try? | ||
==Part II. Electronic vs Biological Circuits== | ==Part II. Electronic vs Biological Circuits== |
Revision as of 10:36, 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:
IntroductionIf you have not had a lesson on how the bacterial photography system works, go read the first half of the design assignment page. Part 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
Building 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... Simulating 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. 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 catalytic efficiency 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 your teacher specifies, 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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