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 define your experimental protocol. (For an outline and overview of the user for experimental protocols go to Experimental Protocols.)

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
• Intravenous infusion of an endogenous chemical substance
• Controlled feeding via an intragastric tube

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

• 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)
• Infusion of 13C-oleate to trace lipid metabolism

To build a tracer protocol you first determine if the tracer and tracer molecule you used are already defined in ProcessDB. From the Molecules and Complexes tab, select the molecule you wish to have traced. (See screen shot). If there are already defined tracers for this molecule, they will be visible at the bottom the tree structure for the traced molecule. Tracer molecules are listed below the list of models that include the traced molecule. Notice that only molecules, not complexes, can be assigned tracers.

To tag a molecule with a new tracer, select the molecule in the database pane and right click on it. If you need a tracer no previous user has entered, select "New Tracer Molecule" from the context menu. You will see the Add Tracer Molecule dialog box. Enter a name for the tracer molecule if desired, or leave it blank if you want ProcessDB to create a default name. The default name is created automatically by inserting an underscore between the molecule name and the tracer name. Select an existing tracer from the Tracer pulldown list. If the tracer does not already exist, select "Add new tracer" and enter a new tracer name in the dialog box. That’s all you need to define a tracer and a tracer molecule.

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.

For the purposes of this 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:

• In the Search box at the top of the Molecules and Complexes tab, start typing the first characters of the molecule name. 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, select it and right click to bring up the context menu. Next you need to select the tracer that you used in your experiment to trace this molecule.

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 create a new Experiment. Click on the Experiments tab, then click on New Experiment icon. Type a descriptive name in the Name field. This opens the screen shown here:

Tip: If you already know the model for which this experiment is designed, you can make Experiment definition easier by selecting the Model in the database pane and dragging it to the “Target Model” field. Doing so allows you to pick from a list any of the states and processes in the Target Model when defining your protocol. This is so easy that we all use this tip.

Your Experiment will be given a unique ID. Since this is a tracer protocol, click the Tracer Protocols tab, and see the following screen:

From the Molecules and Complexes tab, select the Sec7_RFP tracer molecule and drag it to the Tracer Protocols area to the right. This will associate the tagged molecule with the experiment. Click on this molecule so that the name and ID are highlighted in order to use it in the next step.

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 by selecting the Bolus Inputs tab, and biosynthesis can be specified in the Infusion/Biosynthesis tab. For tracers based on fluorescent protein tags it iss 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. In the Infusion/Biosysnthesis tab, click on the "+" at the bottom of the screen. You will see a window with the available states from the target model. Select the Sec7 in cytoplasm state and click on OK. Enter the rate, start and end times. Click on the Save button on the toolbar on the top left of the screen to save your work. Filled out to this point the Experiment panel will look like this:

Finally you need to specify the state (or states) that are photobleached to initiate the FRAP experiment. With the tagged molecule name still highlighted, click on the Photobleaching tab. Click on the "+" in the bottom of the tab, 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). 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.

Remember to click on the save button to save your work as you add and update information on this screen.

You are now ready to link experimental data to the model and to run your protocol on the model, Step 4 and Step 5 respectively.