Picture This

Computer-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 TinkerCell

TinkerCell can be downloaded for free. 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 (1.2.472). If you are running a different version of TinkerCell or 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

• the Molecules and Reaction tabs: try to select 2 molecules from the molecules that are available. For example, click the “Enzyme” on the icon strip and then click the network canvas to place an enzyme. Repeat with a second molecule, selecting “Transcription Factor” from the icon strip and placing it on the network canvas. Next, choose the Reaction tab and select either activation or repression. Click on the enzyme first, then the transcription factor. If you chose activation, you’ll be asked to choose between two mechanisms. A reaction arrow should appear. Next, if you like, try stamping out two receptor molecules and connect them with a different kind of regulation, or try making an enzyme catalyze a reaction with one or more small molecules.
• the Parts and Regulation tabs: Choose the Parts tab and try stamping out a gene expression cassette, i.e. an operator (activator or repressor binding site), a promoter, an RBS, a protein coding sequence, and a terminator (optional). 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. You can then choose “Transcription Regulation” from the Regulation tab, and link the transcription factor you placed earlier to the gene’s operator. A reaction arrow should appear. You can move the icons on the canvas, reshape the reaction arrows, and relabel the parts to your liking. Try using “Protein Production” from the Reaction tab to make the coding region of the gene produce a protein.

Building the bacterial photography system

Now that you have the basic mechanics in hand, you can build the bacterial photography system. Follow the steps below.

• • Start this project on a new canvas. Select “New Canvas” from the File Menu, or click the new page icon on the top toolbar.
  • Assemble the reporter gene: From the “Parts” tab, place an “Activator Binding Site”, “Promoter,” “RBS,” and “Coding” icon on your canvas. Drag the parts next to each other, in that order, so they snap together.
  • Name the reporter gene elements: Click on the name below each part to rename it. The promoter should be named “PompC.” The RBS can be left as is. The coding sequence can be renamed “LacZ”.
  • Add the transcription factor: From the “Molecules” tab, select “Transcription Factor” and place one on 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 choose “Add decorator.” A dialog box will display a choice of icons. From the “Decorators” tab, select “phosphorylation”.

• Activate Transcription of PompC with OmpRp: From the “Regulation” tab, choose “Transcriptional Activation” and then click on OmpRp and the activator binding site just before PompC. Choose Transcription Activation from the pop-up menu.
• 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.
• 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 a Chassis: From the “Compartments” tab, choose Cell and place one on the canvas. Move and resize the cell so it encloses the transcription factor and the reporter gene. Leave the Cph8 receptor in the cell membrane.
• Add light: From the “Molecules” tab, choose “Small Molecule” and print one on the canvas. Rename it “Light” and connect it to Cph8 with an activation arrow from the “Reaction” tab.
• Turn the light on: From the “Inputs” tab, choose “Step input” and click on the Light molecule. Next, find the cell compartment in the “Model Summary” list of components on the right. Click the arrow to expand its subcomponents, find the Light molecule, and expand it. Change the value of “step_time” to 50 (this will turn on the light halfway through the simulation, rather than right at the beginning).

Whew! Now we’ll go on to simulate the photography system in action…

Simulating the bacterial photography 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 make the output a little easier to read, 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 take time now to answer Questions 1-3 found in the “Putting it all together” section (below), instead of waiting until the end of the exercise.

Tuning the system

Turn to the slider window and start changing numbers, making notes of what effects you see. Remember that if the system’s behavior gets way out of whack, 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.

SUGGESTIONS FOR TWEAKING THE SYSTEM

You may be asked to run these simulations, to take notes on your findings, or to skip ahead to the next section, called “Let the games begin.”

• 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?

LET THE GAMES BEGIN

Next, use this model to realize a design specification you have in mind. First, you must decide on some specific changes you would like to make to the system. Would you like the bacterial photographs to develop faster/slower/darker/lighter…? Next, think about how that change would affect the simulation and the graph. Finally, see if you can find the right combination of sliders to make those changes happen. Here are some ideas:

• the bacterial photographs require at least 24 hours to develop in the lab, but they are stable once formed. What elements in the system could be changed to make the COLOR output accumulate more slowly? More quickly?
• the bacterial photographs can sometimes look less intensely colored than the original mask that’s used to generate the image. What elements in the system could be varied to change the total amount of color produced?

Putting it all together

Once you’ve worked through some simulations (or while you work through them), you may be asked by your teacher to answer some of the following questions.

1. What are some approximations/simplifications that you made as you built the model? For example, if you followed these directions then there is no phosphorylated form of Cph8. Do the approximations you made matter?
2. 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.)
3. 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)?
4. Choose at least two adjustments that you made to the sliders and describe
   • what change you made
   • how that change affected the shape of the lines on the graph, and
   • why each adjustment made the difference that it did.
5. Describe how you might carry out some of the simulated adjustments in the lab or in a real cell.
6. Why is it valuable for scientists to be able to model their experiments? What about the value of models and simulations for engineers? Finally, did you personally find it valuable (why/why not)?
7. What are some of the drawbacks to 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?
8. 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 submit them to the TinkerCell development team.
9. Finally, did this exercise suggest any modifications you would like to make to the system, or experiments you would like to try?

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

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

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: a light sensor. When light shines on the photodiode, its resistance decreases, and current flows through it. The photodiode is analogous to the Cph8-OmpR signaling system in the living bacterial photography system.
• OpAmp: a signal propagator. 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. The OpAmp is analogous to the transcription/translation machinery that translates an OmpR signal into synthesis of LacZ in the living bacterial photography system.
• 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 in the living bacterial photography system.
• 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. The LED is analogous to LacZ in the living bacterial photography system.

Introduction 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.

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

1. 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. To get a good connection, make sure you are clamping down on a metal wire and not on the plastic insulation around each wire.
2. Now plug in the LED and the resistor. Push in the wires firmly to make a good connection.
3. You should see light. If not, check your connections, make sure your battery is not dead, etc.

A few hints:

• Remember that the LED allows current to flow through it in only 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.

Building the bacterial photography 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.)
    • Get familiar with the OpAmp’s configuration

    • 

  This image is reproduced from the OpAmp’s

data sheet

 and shows the pins numbered relative to the small “dot” in the OpAmp’s upper corner.
• NC = pin not connected to anything
• -IN = negative input
• +IN = positive input
• -Vs = negative voltage supply = ground
• +Vs = positive voltage supply = +9V
• OUT = output

1. 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).
2. Connect the OpAmp’s plus input (pin 3) to ground.
3. 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.
4. 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.
5. 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).
6. Double check your connections with the circuit diagram above before you power it up.
7. Test your circuit by shining a light on the photodiode and seeing if the LED responds.

Putting it all together

1. Explain the role each component plays in the electronic circuit. Which part of the biological circuit does each represent?
2. No doubt, building this circuit was quicker and easier than actually assembling a biological circuit — for example, you didn’t have to wait for bacteria to grow, or spend time cutting and pasting wires the way you would cut and paste DNA to make a functional plasmid. What are some traits of these components, of the breadboard, or of electronics in general, that make electrical engineering easy in this way?
3. Every analogy has limitations. What are the limitations of the circuit you built as a model of the bacterial photography system? What could you do to make it more realistic?

Examining system behavior at different gain values

If your teacher specifies, continue to this exercise for exploring gain. You may be asked to answer the questions associated with each section.

INITIAL CONDITION: RESISTANCE = 10 MΩ

Right now, the OpAmp’s output and minus input are connected with a 10 MΩ 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 get the LED to hold steady at 1/2 its maximal brightness, by moving the flashlight farther away, shading it, etc?
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?

MODIFICATION: RESISTANCE = 0Ω

Replace the 10MΩ resistor 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 get the LED to hold steady at 1/2 its maximal brightness?
4. Add a line for this circuit to your graph. Is this circuit’s behavior better described as a switch or a dial?

ALTERNATIVE: RESISTANCE = INFINITE Ω

Remove the wire connecting the OpAmp’s output to its negative input.

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 get the LED to hold steady at 1/2 its maximal brightness?
4. Add one last line to your graph. Is this circuit’s behavior better described as a switch or a dial?

Putting it all together

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. More precisely, the gain is the slope of the LED-output-vs.-photodiode-input line. Because the maximum brightness is the same for every circuit, a high-gain amplifier will cause the LED’s brightness to max out even at a low level of input to the photodiode, whereas a low-gain amplifier will cause the LED’s brightness to increase slowly before maxing out, as the photodiode input increases. We can tune this gain by changing the value of the gain resistor.

1. How does this relate to the end of the computer simulation exercise, when you tuned the system with sliders?
2. When might you want switch-like behavior in a biological system, and when might you want dial-like behavior?
3. What are some everyday examples of switch-like and dial-like systems? What element or aspect plays the role of “gain” in each one?

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 BioBuilder 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.