Rewrite instructions for current modeling procedure to reflect changes in TinkerCell interface
Add extensions at end for further realism
LacZ protein production
Catalysis of reporter molecule
Degradation of reporter molecule
(Realistic phosphorylation/dephosphorylation of Cph8 and OmpR? too advanced? more appropriate for college)
In graph section, give qualitative overview of kinetics, no diffeqs
Discussion questions: Explain what's happening in the graph, why each line has the shape it does. What happens if you change this slider and why?
Talk about gain?
On teacher side, link to a good kinetics primer with diffeqs in it
Prepare .tic files with simulation fully built and make available for download (where?)
Ask Deepak why do dependent variables appear in the slider box (or what am I misunderstanding)
Send bugs to Deepak
OTHER TODO:
Move circuit gain exercise to a separate page as it is optional and kind of long; talk less about gain in system design and building-the-circuit sections
Format page nicely
Lab 3: Picture this
Explore 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
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, gain, tuning
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.
Introduction
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
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."
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 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 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. Adding visual appeal
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).
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
Simulation
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?
particularly useful slidersoutput of simulation
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
Circuit kit.
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
Safety 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:
Never directly connect the two battery terminals (+/power and -/ground) with only wire. This is called a "short circuit" and can damage or drain your battery.
Take note of which components require power to be applied in one direction and not the other. Applying power backwards can fry your components. Specifically:
The OpAmp has one pin for + power and one pin for ground.
The diodes (photodiode and LED) must have their positive legs toward power and their negative legs toward ground. Notice that the diodes are round except for one flat side, which indicates the negative leg (flat line looks like a minus sign).
Make sure not to connect your battery backwards. Use red for power and black/blue for ground.
Wires and resistors don't care which direction they are plugged in.
System design
A diagram of the electrical circuit that is analogous to the bacterial photography system. Circuit designed by Steve Wasserman, MIT
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:
Photodiode: #LT959X-91-0125: a light sensor (analogous to the Cph8-OmpR signaling system). When light shines on the photodiode, its resistance decreases, and current flows through it.
OpAmp: #AD8031ANZ: 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.
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.)
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.)
Move part numbers to teachers notes only.
Photodiode (sensor)
OpAmp (logic)
Resistor (gain)
LED (actuator)
Intro to Breadboards
You 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.
Example showing breadboard connections.
For example:
The purple wire and the orange wire are connected. The orange wire bridges the two separate halves of the breadboard.
The purple wire and the green wire are not connected.
Power is delivered to the red rail at the top of the breadboard via the red binding post, which connects to the battery holder. The short yellow wire, the purple wire, and the orange wire are all connected to power.
Ground is delivered to the blue rail at the bottom of the breadboard via the black binding post, which connects to the battery holder. The green wire is connected to ground.
Hooking up an LED
Our basic "Hello, World!" circuit.
Remember 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
Finished circuit.
Power the breadboard: Connect the battery leads to wires using the binding posts. Connect the power wire to the red rail at the top of the breadboard, and the ground wire to the blue rail at the bottom. (Power off the breadboard before you continue building, by disconnecting the rails or snapping the battery out of its holder.)
Position the OpAmp across the trench in the breadboard.
Note the small dot or half-circle marking pin 1 or the "top" of the OpAmp, as shown in the connection diagram. Power the OpAmp by connecting its voltage source inputs to the power rails (pin 7 to +9V, pin 4 to ground).
Connect the OpAmp's plus input (pin 3) to ground.
Diagram showing the connection of each pin on the OpAmp. -IN = negative input, +IN = positive input, -Vs = negative voltage supply = ground, +Vs = positive voltage supply = +9V, OUT = output, NC = pin not connected to anything. (Reproduced from data sheet.)
Connect the OpAmp's minus input (pin 2) to the photodiode. Remember, the photodiode is polarized. It must be connected so that the leg under the flat side is to ground.
You'll make 2 connections to the OpAmp's output (pin 6). First, connect it to the LED and the small, 820 ohm resistor. Remember, the LED is polarized. It must be connected so that the leg under the flat side is to the resistor and the other leg is to power.
Next, connect the OpAmp's output (pin 6) to the large, 10 mega-ohm resistor. Connect the other end of the resistor to the OpAmp's minus input (pin 2). You will change the gain of the amplifier by varying the resistance at this point (using the large resistor, a wire for zero resistance, and no wire for infinite resistance).
Double check your connections with the circuit diagram above before you power it up.
Test your circuit by shining a light on the photodiode and seeing if the LED responds.
Examining the system behavior
Resistance = 10 MΩ
Right now, the OpAmp's output and minus input are connected with a 10 MΩ resistor.
What happens to the LED when you power up the circuit?
What happens to the LED when you shine the flashlight on the photodiode?
Can you get the LED to hold steady at 1/2 its maximal brightness, by moving the flashlight farther away, shading it, etc?
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 = 0Ω
Replace the 10MΩ resistor with a wire.
What happens to the LED when you power up the circuit?
What happens to the LED when you shine the flashlight on the photodiode?
Can you get the LED to hold steady at 1/2 its maximal brightness?
Add a line for this circuit to your graph. Is this circuit's behavior better described as a switch or a dial?
Resistance = infinite Ω
Remove the wire connecting the OpAmp's output to its negative input.
What happens to the LED when you power up the circuit?
What happens to the LED when you shine the flashlight on the photodiode?
Can you get the LED to hold steady at 1/2 its maximal brightness?
Add one last line to your graph. Is this circuit's behavior better described as a switch or a dial?
Discussion
Every analogy has limitations. What are the limitations of the circuit you built as a model of the bacterial photography system?
When might you want switch-like behavior in a system, and when might you want dial-like behavior?
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.
