BioBuilding: Synthetic Biology for Students: Lab 3: Difference between revisions
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* Explain how synthetic biology as an engineering discipline differs from genetic engineering. | * Explain how synthetic biology as an engineering discipline differs from genetic engineering. | ||
* Define and properly use synthetic biology terms: system, | * Define and properly use synthetic biology terms: system, | ||
* Define and properly use molecular genetics terms: two component system, | * 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. | ||
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====Instructions to visually build the bacterial photography system==== | ====Instructions to visually build the bacterial photography system==== | ||
Now that you have some of the basic mechanics in hand, try to visually construct the bacterial photography system according to the tutorial linked [[Media:TinkerCellTutorial.pdf| here.]] | Now that you have some of the basic mechanics in hand, try to visually construct the bacterial photography system according to the tutorial linked [[Media:TinkerCellTutorial.pdf| here.]] | ||
* | *'''Start this project''' on a new canvas, that you can select from the File Menu as a "new canvas" or you can use the icon at the top of the page on the left. | ||
*'''Assemble reporter gene | *'''Assemble reporter gene''': From the “Parts” tab, place an “Inducible Promoter,” “RBS,” and “Coding” icon on your canvas. | ||
*'''Align and name report gene elements | *'''Align and name report gene elements''': Drag items on the canvas next to one another to align them. They’ll turn red when they are connected. Finally, click on the name that’s below the parts to rename each one. If you click on the icon, a dialog box that you don’t want yet may appear. You can just close it and try to click on the name below each icon. The promoter should be named “PompC.” The RBS can be left as is. The coding sequence can be renamed “LacZ.” | ||
*'''Add transcription factors | *'''Add transcription factors''': From the “Molecules” tab at the top of the program, select transcription factor, and print one onto the canvas. It will represent the phosphorylated form of OmpR, so rename it OmpRp. | ||
*'''Visual appeal | *'''Visual appeal''': Select the OmpRp protein and then from the “Edit” menu at the very top of the screen, choose “Add decorator.” A pop-up screen (shown on the right here) will allow you to “replace” the OmpRp with a phosphorylated icon. If you can’t find the phosphorylation icon, make sure you are on the “decorator” tab in the pop-up menu. | ||
*'''Activate Transcription of PompC with OmpRp''': From the “Regulation” menu, select the regulation icon and then click on OmpRp and the pOmpC box. Choose “Transcriptional Activation” from the pop-up menu (shown here). | |||
*''' | *'''Add the Cph8 light receptor''': From the “Molecules” tab, choose “Receptor” and print one on the canvas. Rename it Cph8. | ||
*'''Add | *'''Regulate OmpRp with Cph8''': From the “Regulation” tab, choose “regulation” and then click on Cph8 and OmpRp. Choose Allosteric Inhibition from the pop-up menu. In its nonphosphorylated form, Cph8 inhibits the activity of OmpRp. You can reshape the regulatory arrows and move the elements around the canvas as needed for clarity. | ||
*''' | *'''Add a Chassis''': From the “Compartments” tab, choose “Cell” and print one on the canvas. Select the cell then move and resize it so it encases the transcription factors and reporter construct. Leave the Cph8 receptor in the membrane of the cell. | ||
*'''Add | *'''Add light''': From the “Molecules” tab, choose “small molecule” for the canvas. Rename it “light” and connect it to Cph8 with an activation regulatory arrow. This arrow can be placed by selecting “Regulation” from the “Regulation” tab, clicking on the light and the Cph8 icons and then choosing “Activation” from the popup menu. | ||
*''' | |||
====Whew!==== | ====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? 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? | 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? <br> | ||
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 system==== | |||
===Part II. Electronic vs Biological Circuits=== | ===Part II. Electronic vs Biological Circuits=== | ||
In this activity, we'll explore signalling 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.[[Image:ElectronicsKit pic.jpg|thumb|left|200px|'''starting kit''']] | In this activity, we'll explore signalling 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.[[Image:ElectronicsKit pic.jpg|thumb|left|200px|'''starting kit''']] |
Revision as of 11:42, 21 January 2011
Eau That Smell Lab |
Lab 3: Picture this
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 WassermanObjectivesBy 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? Instructions to model the bacterial photography systemPart II. Electronic vs Biological CircuitsIn this activity, we'll explore signalling 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.Safety informationIn this exercise, you'll be working with circuits connected to a battery. Practice good habits by never touching the circuit without first unplugging it.
When you connect the battery to the breadboard, make sure you do so in the correct orientation for the circuit (red to red, black to ground) or the OpAmp will break...really.
System designThis system is fairly simple one since it only consists of 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. As you can see in the schematic, the circuit contains the following parts.
Let's start buildingThe circuit has been constructed using a breadboard which is a convenient way to construct electrical circuits. The breadboard holes are connected beneath the plastic as shown in the photo. Take note of these connections because they'll affect how you will connect up components in this exercise.
Examining the system behaviorResistance = infinite ΩAir connects the OpAmp's pin 6 to pin 2
Resistance = 0ΩConnect the OpAmp's pin 6 to pin 2 with a wire.
Variable resistanceConnect the OpAmp's pin 6 to pin 2 with a 10 MΩ resistor.
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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