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| [[PDF of this page]] | | [[Media:BioBuilding Students Lab3.pdf| PDF of this page]] |
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| '''TINKERCELL TODO:'''
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| * Retake screenshots
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| ** On teacher side, link to a good kinetics primer with diffeqs in it
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| * Prepare .tic files with simulation fully built and make available for download (where?)
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| * Send bugs to Deepak
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| '''OTHER TODO:'''
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| * 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
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| * Format page nicely
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| =Lab 3: Picture this= | | =Lab 3: Picture this= |
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| Explore an engineered biological system through a computer simulation, an electronics building kit, and a real-life example. | | Explore an engineered biological system through a computer simulation, an electronics building kit, and a real-life example. |
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| [[Image:PictureThis.png]] | | [[Image:Lab3 photoheader.png|600px]] |
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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''' | | '''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''' |
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| ==Objectives== | | ==Objectives== |
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| * Define and properly use molecular genetics terms: two component system, transcriptional activation, phosphorylation | | * 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. |
| | * Explain the role that modeling can play in design, and name some ways that models differ from reality. |
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| ==Introduction== | | ==Introduction== |
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| ==Part I: TinkerCell==
| | 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]]. |
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| 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.
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| [[Image:TInkerCell.png|thumb|left| [http://www.tinkercell.com TinkerCell] ]]
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| One early effort at a CAD tool for synthetic biology is [http://www.tinkercell.com 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 [http://www.jbioleng.org/content/3/1/19 this article] from the Journal of Biological Engineering.
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| ===Getting Started with TinkerCell===
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| TinkerCell can be downloaded for free from [http://www.tinkercell.com/downloads-2#TOC-Download-current-versions 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.
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| After you open the TinkerCell application, begin familiarizing yourself with the basic operation of the program. In particular, try to use
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| * 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.
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| * 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.
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| ===Instructions to build the bacterial photography system===
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| Now 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 [[Media:TinkerCellTutorial.pdf|here.]] ''Do not actually use the additional tutorial, it is not updated''
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| *'''Start this project''' on a new canvas. Select "New Canvas" from the File Menu, or click the new page icon on the top toolbar.
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| *'''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.
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| *'''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".
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| *'''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".
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| *'''Visual appeal''': Select the OmpRp protein, and then from the "Edit" menu choose "Add decorator." A pop-up screen (shown on the right here) will display a choice of icons. From the "Decorators" tab, select "phosphorylation".
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| [[Image:Add decorator.png|thumb|250px|right|Adding visual appeal]]
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| *'''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 (shown here).
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| [[Image:AddTxnReg.png|thumb|right|Regulating transcription]]
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| *'''Add the Cph8 light receptor''': From the "Molecules" tab, choose "Receptor" and place one on the canvas. Rename it "Cph8".
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| *'''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.
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| ** ''(In reality, in its nonphosphorylated form, Cph8 inhibits the activity of OmpRp. Thus, "phosphorylation-dephosphorylation cycle might be a better representation of reality, but "allosteric inhibition" works for our purposes and is simpler.)''
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| *'''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.
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| *'''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.
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| *'''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.
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| *'''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.
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| *'''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).
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| Whew! Now we'll go on to simulate the photography system in action...
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| ===Instructions to simulate the bacterial photography system===
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| [[Image:DeterministicSimulation.png|thumb|300 px|Simulation ''(change)'']]
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| 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.
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| [[Image:TinkerCell ModelOutput.png|thumb|300px|right|output of simulation]]
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| 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. ''(Everything else is either constant or an internal TinkerCell bookkeeping variable.)'' Take a few minutes to familiarize yourself with the output graph. What are the axes? ''(Arbitrary units.)'' Which lines go up and which go down when the light turns on? How fast? Do any lines stay the same when the light turns on? Why? ''The enzyme degrades but the small molecule doesn't.''
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| ====Tuning the system====
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| 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 catalysis power of the BetaGal enzyme.
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| Now try to achieve some specific goals by changing the sliders. For example, in reality, a bacterial photo takes some time to develop, but is stable once it's formed. Can you make the COLOR output accumulate more slowly? More quickly? Or, try changing the light's step input to a sine wave input, and change the frequency. Can you make the amount of BetaGal protein go up and down?
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| [[Image:Tinkercell especiallyUsefulSliders.png|thumb|300px|left|particularly useful sliders]] | |
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| 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?
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| ===Putting it all together===
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| 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, like 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?
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| If you'd like, you can go on to use the TinkerCell program to model the answers to some of these questions....
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| 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.
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| ==Part II. Electronic vs Biological Circuits==
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| [[Image:ElectronicsKit pic.jpg|thumb|right|200px|'''Circuit kit.''']]
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| 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.
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| ===Safety===
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| '''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.
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| '''Safety for the circuit components:'''
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| * 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.
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| * 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:
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| ** The OpAmp has one pin for + power and one pin for ground.
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| ** 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).
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| ** Make sure not to connect your battery backwards. Use red for power and black/blue for ground.
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| ** Wires and resistors don't care which direction they are plugged in.
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| <br style="clear:both" />
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| ===System design===
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| [[Image:Systemdesign.png|thumb|center|450px|'''A diagram of the electrical circuit that is analogous to the bacterial photography system.''' Circuit designed by Steve Wasserman, MIT]]
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| 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:
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| #[[Media:Photodiode SpecSheet.pdf| 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. <br>
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| #[[Media:OpAmpDataSheet.pdf| 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.<br>
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| #[http://www.breakup.de/resources/resistor.html 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.)''<br>
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| #[http://www.allelectronics.com/make-a-store/item/LED-2/GREEN-5MM-T1-3/4-LED//1.html 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.)<br>
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| ''Move part numbers to teachers notes only.''
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| <table border="1">
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| <tr>
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| <td>[[Image:Photodiode.jpg|150px|Photodiode (sensor)]]</td>
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| <td>[[Image:Opamp.jpg|150px|OpAmp (logic)]]</td>
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| <td>[[Image:Bigresistor.jpg|150px|Resistor (gain)]]</td>
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| <td>[[Image:Ledresistor.JPG|150px|LED (actuator)]]</td>
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| </tr>
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| <tr>
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| <td>Photodiode (sensor)</td>
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| <td>OpAmp (logic)</td>
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| <td>Resistor (gain)</td>
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| <td>LED (actuator)</td>
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| </tr>
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| </table>
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| ===Intro to Breadboards===
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| 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.)
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| 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.
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| [[Image:Breadboard.jpg|thumb|left|300px|Example showing breadboard connections.]]
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| For example:
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| * The purple wire and the orange wire are connected. The orange wire bridges the two separate halves of the breadboard.
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| * The purple wire and the green wire are '''not''' connected.
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| * 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.
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| * 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.
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| =====Hooking up an LED=====
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| [[Image:Helloworld.png|thumb|right|250px|Our basic "Hello, World!" circuit.]]
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| 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!)
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| 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.
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| 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.)
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| 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.
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| ===Let's start building===
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| [[Image:Finishedcircuit.jpg|thumb|right|200px|'''Finished circuit.''']]
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| #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.)
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| #Position the OpAmp across the trench in the breadboard.
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| #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).
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| #Connect the OpAmp's plus input (pin 3) to ground.
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| [[Image:AD8031pinouts.png|thumb|right|200px|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 [[Media:OpAmpDataSheet.pdf|data sheet]].)]]
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| #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.
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| #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.
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| # 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).
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| #Double check your connections with the circuit diagram above before you power it up.
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| #Test your circuit by shining a light on the photodiode and seeing if the LED responds.
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| ===Examining the system behavior===
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| =====Resistance = 10 MΩ=====
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| Right now, the OpAmp's output and minus input are connected with a 10 MΩ resistor.
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| #What happens to the LED when you power up the circuit?
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| #What happens to the LED when you shine the flashlight on the photodiode?
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| #Can you get the LED to hold steady at 1/2 its maximal brightness, by moving the flashlight farther away, shading it, etc?
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| 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.
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| 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.
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| 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?
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| =====Resistance = 0Ω=====
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| Replace the 10MΩ resistor with a wire.
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| #What happens to the LED when you power up the circuit?
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| #What happens to the LED when you shine the flashlight on the photodiode?
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| #Can you get the LED to hold steady at 1/2 its maximal brightness?
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| #Add a line for this circuit to your graph. Is this circuit's behavior better described as a switch or a dial?
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| =====Resistance = infinite Ω =====
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| Remove the wire connecting the OpAmp's output to its negative input.
| | ==[[BioBuilding: Synthetic Biology for Students: Lab 3 --Tinkercell| Part I: TinkerCell]]== |
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| #What happens to the LED when you power up the circuit?
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| #What happens to the LED when you shine the flashlight on the photodiode?
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| #Can you get the LED to hold steady at 1/2 its maximal brightness?
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| #Add one last line to your graph. Is this circuit's behavior better described as a switch or a dial?
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| ===Discussion===
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| * Every analogy has limitations. What are the limitations of the circuit you built as a model of the bacterial photography system?
| | ==[[BioBuilding: Synthetic Biology for Students: Lab 3 --Electronics| Part II. Electronic vs Biological Circuits]]== |
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| * When might you want switch-like behavior in a system, and when might you want dial-like behavior?
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| * How does this relate to the end of the computer modeling exercise, where you tuned the system by changing the sliders?
| | ==[[BioBuilding: Synthetic Biology for Students: Lab 3 --U-do-it bacterial photograph| Part III. U-do-it Bacterial Photograph]]== |
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| ==Part III. U-do-it Bacterial Photograph== | |
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| 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.
| | ==Data Sharing== |
| | When you've finished your work on any part of this activity, upload your data to the link on the BioBuilder site that's [http://www.biobuilder-submitdata.org/users/login here.] You'll be able to compare what you've done to what other BioBuilders around the country have tried. |
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| 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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| ==Navigation== | | ==Navigation== |
