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STEP 3: Record Your Experimental Protocols
ProcessDB handles a wide variety of experimental protocols and automatically applies them to the model you choose. In order for us to do this accurately you have to fill in a form that defines your experimental protocol.
ProcessDB experimental protocols fall in three broad categories, and each will be described in detail as we take you through Step 3. The three categories are
State protocols
Process protocols
Tracer protocols
State protocols are needed when your experiment involves experimenter-controlled state variables. Examples in a cell culture experiment are
Replacing the medium with medium of different chemical composition
Addition of a hormone or agonist to the medium
Addition of a drug or inhibitor to the medium
Microinjection of a chemical substance into the cytoplasm
Imposition of a change in some physical force such as temperature or membrane potential
Process protocols are needed when your experiment involves experimenter-control of the rate at which some molecule is added or removed. These are the least common protocols, but they are very common in some scientific fields. Examples are
Intravenous infusion of an endogenous chemical substance
Controlled feeding via an intragastric tube
Opening a Guytonian arterio-venous shunt
Controlled hemorrhage
Controlled perfusion rate in an isolated organ preparation
Controlled perfusion of a tissue or cell culture chamber
Controlled flows in microfluidic labs-on-chips
Tracer Protocols are needed in a wide variety of biochemical, cell biological, and physiological experiments. The original idea of tracer molecules was to capture dynamic information from steady state systems, but modern applications have expanded the utility of tracers in many fields. Examples are
Bolus intravenous injection of 14C-labeled substrates or precursors to follow metabolism
Pipetting 35S-methionine into the medium of a cell culture to trace protein synthesis
Adding AT32P to a cuvette containing isolated kinases, phosphatases, and protein substrates
Transfection of a genetic construct encoding a fusion protein with GFP
Fluorescence recovery after photobleach (FRAP)
Fluorescence loss in photobleaching (FLIP)
Inverse FRAP (iFRAP)
Photoactivation of a genetically encoded fluorescent protein (e.g. PA-GFP)
To build a tracer protocol you first determine if the tracer you used is already defined in ProcessDB. From the main Forms Menu click Experiments/Define Tracers. (See screen shot). This opens the form where you can see the already defined tracers and add any you may need. Immediately click Query/Execute (or the Execute Query button on the toolbar) to see tracers already defined. (See screen shot)
If you need a tracer no previous user has entered in the table, just type its name on the first empty row in the Tracer Name column and click Action/Save or the Save icon on the toolbar. That’s all you need to define a tracer. Click Action/Exit or the Exit icon on the toolbar to return to the main ProcessDB menu.
Because there are many available tracers and many molecules you might want to trace, ProcessDB allows you to combine any tracer with any already-defined molecule to yield a useful tracer molecule. In general you will already have defined all the molecules you need in Step 1. Sometimes, however, you will need to go back to Diagrams and add a molecule you have forgotten. That’s no problem in ProcessDB. You are always free to move forward and back in the ProcessDB modeling process.
If the molecule you want to trace is already in ProcessDB, you are ready to create a tracer molecule. On the main Forms menu click Experiments/Define Tracer Molecules. This opens a form when any tracer can be combined with any molecule. (See Screen Shot)
Like most ProcessDB forms this form opens in query mode ready to retrieve an existing tracer molecule. If you want to see what some look like before you create your own, just click Query/Execute or the Execute Query button on the toolbar. (See screen shot)
To define a new tracer molecule, clear the form by clicking Block/Clear, click the first blank row under Molecule Id, and then click Edit/Display List to bring up the list of available molecules. You can also bring up this list by typing Ctrl-L after you click in the first blank row under molecule ID. Of course if you have a great memory you can just type in a remembered molecule ID and press Tab. It’s never a requirement to remember IDs, but they often save keystrokes.
This list has the jargon name "List of Values" (LOV). For the purposes of this tutorial/user guide we need a new tracer molecule, catalytic subunit of cAPK tagged with RFP (red fluorescent protein). There are two ways to find a molecule that’s in the ProcessDB database:
When the LOV window opens, just start typing the name of the molecule. You can use the Oracle wildcard % to represent any number of characters before or after a string of text. The list of molecules is then automatically narrowed down based on what you type.
You can just scroll through the list (not recommended unless your molecule begins with a number or the letter A).
When you find your molecule, highlight it and click OK. You can also just double-click it if you prefer. Either way, the LOV window will close and your selected molecule will be inserted in the previously blank row. Next you need to select the tracer that you used in your experiment to trace this molecule.
Click in the row where you entered your molecule but in the Tracer ID column. Use your freshly-learned LOV skills to insert your tracer and hit tab. Your tracer molecule will be named automatically by inserting an underscore between the molecule name and the tracer name. Click Edit/Save or the Save icon on the toolbar. That’s it for defining your tracer molecule. If you have more than one tracer molecule, just do the same on succeeding rows of the Define tracer molecules screen. If you’re going to define your tracer protocol right away, you might want to briefly memorize the tracer molecule ID that appears after you Save. This will save keystrokes when you define your Tracer Protocol. Click Action/Exit to return to the main ProcessDB menu.
Tip: Lists of values are available for many fields in ProcessDB Forms. You can tell if you are in a field that supports LOV by looking at the status bar at the bottom of the Forms screen.
You may have to scroll down to see it, but if you look at this screen shot you’ll see "List of values...", which indicates the current field has a LOV available.
Now you are ready to define the experimental protocol you used (or plan to use) to collect your tracer data. As an example, here is a simple model of a yeast cytosolic protein, Sec7, that also associates peripherally with Golgi membranes.
Suppose you have labeled Sec7 with RFP and you want to do a Golgi FRAP experiment. Using the steps outlined above, you would first define the Sec7_RFP tracer molecule, and then click Experiments/Define, Edit and Query Experiments. This opens the screen shown here:
Like most ProcessDB screens, this one opens in Query mode ready to retrieve Experiments you’ve already stored in the ProcessDB database, but now we want to define a new experiment so first click Query/Cancel. Then name your experiment by typing a descriptive name in the Name field.
Tip: If you already know the model ID for which this experiment is designed, you can make Experiment definition easier by entering the Model ID in the “Model to use to filter State LOVs” field.
Click Action/Save and your Experiment will be given a unique ID. Since this is a tracer protocol, click the Tracer Protocols tab, and see the following screen:
In the Tracer Molecules block enter the Sec7_RFP ID and then Tab. This will display the corresponding tracer name so you are sure you have the one you intended.
Next you should define how the tracer is delivered to the biological system. The most common such inputs are bolus injections of a known amount of tracer, and biosynthetic production. A bolus can be specified easily in the Bolus Inputs block, and biosynthesis can be specified in the Infusion/Biosynthesis block. For tracers based on fluorescent protein tags it’s common to transfect cells with a construct encoding the appropriate fusion protein. This is entered as a number of molecules produced per second (if your experiment is using seconds at its time unit). Since Fluorescence measurements are almost always normalized, you need not worry about the absolute value of this fusion protein synthesis rate. We generally set this to 10 molecules/second. The larger the value and the longer the time before the FRAP, the greater will be the level of overexpression. Transfections often begin the night before the experiment, although if you are using a stably transfected cell line, you may choose your start time based only on the need to reach a steady state before the FRAP experiment begins. The following screen shows a Transfection beginning 15 hours (648,000 seconds) before the FRAP. Negative times can be used for convenience so that the FRAP bleach is at time = 0. All times must be entered as all numbers, with no commas. Assuming synthesis of the fusion protein continues through the entire experiment, you can set the End Time to the time when you stopped taking measurements, 600 seconds in this example. ProcessDB needs to know which model State receives the input of Sec7_RFP. Since Sec7 is translated on free ribosomes, the receiving State is Sec7 in Cytoplasm. Filled out to this point the Experiment Form will look like this:
Finally you need to specify the state (or states) that are photobleached to initiate the FRAP experiment. Click in the first row of the Photobleaching block, click Edit/Display List, select the state you wish to bleach, and click OK. Then enter the start time for the bleach (0 in this case) and the Bleach End Time (1.5 in this case). Click Action/Save. Your experiment is complete. It should look something like this:
You can use as many rows in each block as you require to fully define your tracer experiment. If multiple states are bleached, just insert them in succeeding rows of the Photobleaching block. Though uncommon, it is entirely possible to have multiple tracer molecules in the same experiment. To do this you just include multiple rows in the Tracer Molecules block. Select any tracer molecule to display the corresponding rows in the Bolus, Infusion, Photobleaching, and Photoactivation blocks.
If you wish to define a Bolus Input, indicate the number of tracer molecules added (in the Amount field), the time at which they are added (in the @Time field), and the state ID (in the State Id field) where they are to start. If you want this bolus to distribute to its steady state distribution before your FRAP, you can specify a negative time for the bolus and distribution will occur from then until time=0 when the FRAP bleaching pulse starts. This approach is best used when the model is a closed system or is approximately closed because degradation is extremely slow on the time scale of the experiment.
If the tracer experiment also includes State Protocols or Process Protocols, these must be entered as well in order to completely define your experiment.
You are now ready to link experimental data to the model and to run your protocol on the model, steps 4 and 5 respectively.
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