BioBuilding: Synthetic Biology for Students: Lab 3

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PDF of this page =Lab 3: Picture this=
 * Explore an engineered biological system through a computer simulation, an electronics building kit, and a real-life example.

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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=====

Objectives
By the conclusion of this laboratory investigation, the student will be able to:
 * Explain how synthetic biology as an engineering discipline differs from genetic engineering.
 * Define and properly use synthetic biology terms: 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.
 * Model a biological system using electronic parts and a computer program.

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 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 started
The 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.
 * the “Molecules" and "Regulation” tabs: try to select 2 molecules from the molecules that are available, for example click the “transcription factor” from icon strip and then click the network canvas. Repeat with a second molecule, selecting mRNA from the icon strip and placing it on the network canvas. Connect the two molecules with the “Regulation” options (the 4th tab from the left), choosing the regulation icon from the bar just above the network canvas and clicking on the two molecules you placed on the canvas that you’d like to connect. You can decide to connect the two molecules through a repression or an activation mechanism. A reaction arrow should appear if you’ve done this right. If you’d like to try another before moving on, try to stamp out two receptors and connect them with a different kind of regulatory reaction. Once you’ve gotten used to these operations, try the next ones…
 * the “Parts” and “Reaction” tabs: : Choose the Parts tab and try stamping out a gene expression cassette, i.e. a promoter (any variety), an RBS, a protein coding sequence, and if you like: a terminator. 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. They’ll turn red when they are connected. You can then choose a 1-to-1 reaction from the Reaction tab, and connect the coding sequence you just stamped out to the mRNA you placed on the canvas earlier. A reaction arrow should appear. You can move the icons on the canvas as well as reshape the reaction arrows and label the parts with your favorite part name, for example naming the promoter “Plac.”

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.]]
 * 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: From the “Parts” tab, place an “Inducible Promoter,” “RBS,” and “Coding” icon on your canvas.
 * 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: 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: 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. [[Image:Add decorator.png|thumb|250px|right|Adding visual repeal]]
 * 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). [[Image:AddTxnReg.png| thumb|right|regulating transcription]]
 * Add the Cph8 light receptor: From the “Molecules” tab, choose “Receptor” and print one on the canvas. Rename it Cph8.
 * 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 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!
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 system
If 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 together
The 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 Circuits
In 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.

Safety information
In 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. 
 * also please note:

System design
This 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.
 * 1) [[Media:Photodiode SpecSheet.pdf| Photodiode]]: #LT959X-91-0125: a light sensor (mimics Cph8 and the phycobilins). When light shines on the photodiode, its resistance is decreased and current flows through it.
 * 2) [[Media:OpAmpDataSheet.pdf| OpAmp]]: #AD8031ANZ: a logic device that detects a difference in + and - current inputs and amplifies it.
 * 3) Resistor: components which resists current flow by producing a voltage drop across it. The gain in this system is proportional to the resistance, and by varying the resistance into the OpAmp, the sensitivity of the circuit to light can be tuned.
 * 4) LED: a device with a detectable output (mimics LacZ). A voltage drop across its terminals turns the green-colored light on. The 820&Omega; resistor is added to the circuit before the LED to ensure that the voltage drop across the LED isn't too high (which can cause the LED to breakdown).

Let's start building
The 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.
 * 1) Power the breadboard by running a wire from the +V source (= the red terminal) to the + rail (= the red + connections at the "top" of the breadboard).
 * 2) Run the - rail (= the blue - at the "bottom" of the breadboard) to the black, ground terminal.
 * 3) Use a small wire to connect the A-E rails to +9V.
 * 4) Use a small wire to connect the F-J rails to ground.
 * 5) Position the OpAmp across the trench in the breadboard.
 * 6) Power the OpAmp by connecting it to the +9V and ground using the pin diagram that's in the spec sheet and is reproduced here. Note that the small dot on the corner of the OpAmp indicates pin #1.
 * 7) Connect the OpAmp's + input (pin 3) to ground.
 * 8) Connect the OpAmp's - input (pin 2) to the photodiode. NOTE: the photodiode is asymetric and must be inserted into the breadboard so that the leg under the "flat" edge is to ground. Leave some space between the photodiode input and the OpAmp so an additional resistor can be added to the circuit later.
 * 9) You'll make 2 connections to the OpAmp's output (pin 6)
 * 10) *The first connection from the OpAmp's output should be to the 820 ohm resistor and the LED. NOTE: the LED is asymmetric and must be inserted into the breadboard so that the leg under the "flat" edge is connected to the 820 ohm resistor and the round side is inserted into the +9V rail.
 * 11) *The second connection is to a wire that runs to a variable resistor (see the "finished circuit"). You will change the current into the OpAmp by varying the resistance through the circuit this point. You will start by putting no resistor in, where air serves as an infinitely large resistor.
 * 12) Double check your connections with the system diagram above before you power it up.
 * 13) To connect the 9V battery to the terminals, place the snap battery cover on the battery. Attach the alligator clip on the red battery lead to the red terminal of the breadboard, and the alligator clip on the black battery lead to the black terminal.

Resistance = infinite &Omega;
Air connects the OpAmp's pin 6 to pin 2
 * 1) What happens to the LED when you power up the circuit?
 * 2) What happens to the LED when you shine the flashlight on the photodiode?
 * 3) Can you shine the flashlight on the photodiode so that the LED holds steady at 1/2 its maximal light intensity? 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.
 * 4) Sketch a graph that has 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 = 0&Omega;
Connect the OpAmp's pin 6 to pin 2 with a wire.
 * 1) What happens to the LED when you power up the circuit?
 * 2) What happens to the LED when you shine the flashlight on the photodiode?
 * 3) Can you shine the flashlight on the photodiode so that the LED holds steady at 1/2 its maximal light intensity?
 * 4) Add a line to your graph that has flashlight intensity on the x-axis and LED light intensity on the y-axis. With zero resistance in place, is the circuit's behavior better described as a switch or a dial?

Variable resistance
Connect the OpAmp's pin 6 to pin 2 with a 10 M&Omega; resistor.
 * 1) What happens to the LED when you power up the circuit?
 * 2) What happens to the LED when you shine the flashlight on the photodiode?
 * 3) Can you shine the flashlight on the photodiode so that the LED holds steady at 1/2 its maximal light intensity?
 * 4) Add one last line to your graph that has flashlight intensity on the x-axis and LED light intensity on the y-axis. Is this circuit's behavior better described as a switch or a dial?

Part III. U-do-it Bacterial Photograph
Decide 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.

Navigation

 * BioBuilding: Synthetic Biology for Students
 * BioBuilding: Synthetic Biology for Students: Lab 1
 * BioBuilding: Synthetic Biology for Students: Lab 2
 * BioBuilding: Synthetic Biology for Students: Lab 3
 * BioBuilding: Synthetic Biology for Students: Lab 4
 * BioBuilding: Synthetic Biology for Students: Essay
 * BioBuilding: Synthetic Biology for Students: Design Assignment
 * BioBuilding: Synthetic Biology for Students: Glossary
 * [back to biobuilder]

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