CybertoryTM

Molecular Biology Laboratory Simulator

Instructions

©1998, Attotron Biosensor Corporation

Attotron Biosensor Corporation
106 East Adams Street, Suite 210
Carson City, Nevada 89706, USA
www.attotron.com

CONTENTS:

  1. INTRODUCTION
  2. SYSTEM REQUIREMENTS
  3. RUNNING THE PROGRAM
  4. SETTING EXPERIMENTAL PARAMETERS
  5. RESTRICTION DIGESTION
  6. GEL ELECTROPHORESIS
  7. ANALYTICAL TOOLS
  8. BUGS, GLITCHES, AND BIZARRE BEHAVIOR
  9. BETA TESTER AGREEMENT

CYBERTORY MAILING LIST


INTRODUCTION

CybertoryTM is a virtual laboratory simulator for conducting molecular biology experiments. It is currently in a prototype stage, and is intended to be made available only to Beta Testers who have agreed to evaluate it as a teaching tool. If you have not already signed up to be a beta tester, please read the last section of this document for instructions on signing up.

This document is also a prototype, and we would appreciate your feedback on any areas that require clarification. Please direct questions or comments to the Cybertory mailing list (cybertory@attotron.com). All beta testers should be subscribed to this list; for information on subscribing, please send e-mail to : cybertory-request@attotron.com.

Development of this software is supported by Small Business Innovation Research (SBIR) grant 1 R43 RR13645-01 from NCRR to Attotron Biosensor Corporation.

SYSTEM REQUIREMENTS

The program is written entirely in Javascript, and runs inside a window of the Netscape Navigator web browser. It makes extensive use of Dynamic HTML, only available in version 4 and above. It has been successfully tested with version 4.06 of Netscape on Macintosh, and with version 4.5 on Windows 95. It crashes reproducibly at the point of loading the gel when run on Netscape Navigator 4.03 on both Macintosh and Windows95. This prototype will not work in any version of Internet Explorer.

A minimum of 32 megabytes of RAM are recommended, with virtual memory enabled and set to a total of at least 64 megabytes. The program runs on a 75MHz Pentium and Performa 200 MHz PowerPC 603e machines, but performance is noticeably better on a G3 Macintosh or a 200 MHz Pentium or Pentium II. A minimum screen resolution of 800x600 pixels is required to run in the default mode as described below.

RUNNING THE PROGRAM

To start the program, open the file START.HTM in the CYBERTOR directory from within Netscape. This will open a page that has a button which you must click to launch the Cybertory program.

The Cybertory window has two frames. The top frame has the Attotron and Cybertory logos, and a pull down menu from which a simulation module is chosen. The simulation module loads in the bottom (larger) frame.

SETTING EXPERIMENTAL PARAMETERS

The restriction enzyme digestion module opens by default when the behind-the-scenes data loading is finished (this may take a while, especially on a slower machine.) This module contains icons representing reaction tubes, reagent tubes, pipet, waste can, tip box, etc. The first four reagent tubes (on the left side of the screen) are for enzymes, and the bottom row are for DNA samples. To set the experimental parameters (i.e., which restriction enzymes and which DNA sequences are to be used), you must first place an order in the Virtual Catalog simulation module. This is accessed from the simulation module pull down menu.

By scrolling the top window down further, you can see that it actually contains boxes which hold data about the tube contents. This is the way data is passed from one module to another. This should normally be hidden from the user, but the scroll bar is left visible for now for debugging purposes.

The Virtual Catalog has two pages. The first describes the virtual supplies and equipment, including information such as restriction enzyme recognition sites and the sizes of the bands on the molecular weight marker. The second page is the Order Form, which is used to select the enzymes and DNA samples to be used in the experiment. Entering the password "Laguna" allows you to fill out the order form and submit it. Use of this page is password protected, so that the teacher can prevent students from changing the parameters. Teachers who want the students to make the decisions about which enzymes to use can simply give them the password.

An order can specify any four of the 78 available restriction enzymes, and a set of six DNA samples. The default DNA samples are a "Forensic Sample Kit" including one tube of DNA from a "Perpetrator", and five samples from suspects. Students can use restriction fragment length polymorphism analysis to identify which suspects can be excluded from matching the perpetrator's sample.

Rather than using the Forensic Sample Kit, "virtual samples" of custom sequences can be created by choosing "Custom DNA Synthesis" from the order form and pasting the desired sequences in the boxes. Currently, the length limit on custom sequences should be around 4000 characters (i.e., 4kb), which is the Cookie length limit in Netscape. Please note that DNA sample labels should be kept to three or four characters, despite the fact that the space on the order form is much longer, since these labels need to fit over the tubes in the restriction digestion simulator.

Pressing the button at the bottom of the form to send the order brings up a "Thank You" screen while the program sets the specified parameters. These parameters are kept as "Magic Cookies", which will persist on the computer until another order is placed, or until they expire. (The program will not expire, only the cookies. They may be reset by placing another virtual order.) Note that if the program is used on a network, the cookies must be set (i.e., an "order" must be placed) on each machine.

RESTRICTION DIGESTION

Restriction digestion reactions are set up by mixing the ingredients: click on a tube to select it, and its top will pop open and the pipet will magically drift over to put its tip in the tube (though, regrettably, not necessarily in that order, depending on how fast your computer is.) Clicking on the plunger toggles between the up or down positions.

Pulling a sample into the pipet tip should lower the fluid level in the source tube. The fluid will also show in the pipet tip, though you may not see it when the tip is in a tube. To see the contents of the tip, click on the open tube to deselect it: this causes the pipet to move out of the tube to its parking area near the right of the screen. The water tube is unique; it magically has 5 milliliters of water in a 500 microliter tube, so quite a bit of volume must be removed before the level drops. When a sample is placed in a reaction tube, its fluid level should also change: 5 microliters is about the minimum quantity detectable in an empty tube.

Though the program does keep track of how much buffer one places in the reaction tubes, all it really cares about at this point is which DNA samples and which enzymes are in which reaction tubes. The reaction is started by clicking the start button on the timer in front of the waste bin. This should magically pop up an incubator to begin the 37°ree;C incubation. All of the virtual enzymes cut at the same temperature, and they all cut to completion no matter how much enzyme one uses. Impatient users can cut the incubation short by clicking the timer button again, which should say "skip" while the incubation is running.

GEL ELECTROPHORESIS

To run a gel, choose the gel simulation module from the pull down menu in the top frame. The gel is loaded by clicking the Auto Load Gel button. This loads whatever samples were in the 12 reaction tubes in the restriction enzyme digester into the first 12 lanes of the gel. All lanes are loaded with blue sample dye whether they contain samples or not. The time and voltage (10 to 1000 V - the gels never melt) can be set on the power supply, either by clicking the control buttons or by typing into the boxes. Pressing "run" turns on the voltage until the time runs down. If the gel is operating properly, you should see bubbles coming off of the electrodes, and the blue dyes should move into the gel and begin to separate.

To see the DNA, you must click the button to turn on the UV light, and click the wall switch to turn off the room lights. Because virtual time passes quickly, high virtual voltages can be used, and virtual UV light is harmless, feel free to watch the bands separate.

Migration of the bands can be measured by dragging the fluorescent ruler over the gel. Turn on the lights to position it at the bottom of the sample wells, then turn the lights off again to see the bands.

ANALYTICAL TOOLS

The fourth module currently available on the pull down menu is a set of simple analytical tools. These tools are intended to be simple rather than exhaustive, and to insure that users have access to at least the basic tools. If you have better analytical software, there is no reason not to use it. The restriction mapper uses the same 78 enzymes available for the digester. It draws a linear graphical representation of a DNA segment with cut sites for the chosen enzyme marked. The DNA segment is of fixed length, and only the scale changes to reflect input sequences of different lengths. The protein translator converts a sequence from DNA to RNA to protein. Only the standard genetic code is available, and it does not (yet?) do reverse translation.

There is also a "Secret Coder" which translates any written message (or any series of typeable characters excluding the percent (%) sign) into DNA. We hope this may be useful in illustrating the idea that DNA can encode information, without having to first explain what a protein is. Another tool in the same vein is the "Random Homologous Recombiner", which creates random recombinations between two input character stirings. These can be DNA sequences, English sentences, etc. The user enters a window size, and a probability (between 0 and 1) that a recombination will occur between the input sequences if they match in a window of the specified size at any homologous position. By "synthesizing" the resultant sequences into virtual samples, one could do restriction maps of Lincoln's Gettysburg Address, or excerpts from Monica Lewinsky's testimony, or some random recombinant between them; these sorts of things we leave to the imagination of the teachers and/or students.

The last tool is a specialized calculator for setting up PCR master mixes, as described in Horton, R.M., and Stone, R.J.S. PCR master mix volume calculators in Javascript. BioTechniques 24(3) , March, 1998.

Two other programs are included on the CD-ROM in the "AUTHORS" directory. One is a specialized program for writing multiple-choice quizzes on web pages (Horton, R.M. Interactive Internet Instruction: Web Quizzes and a New JavaScript Quizmaker. BioTechniques 23(6):1048-1050, December 1997.), and the other is an authoring program for making image maps for web pages (Horton, R.M., and Russell, M.J. Making client-side image maps. BioTechniques 25(1):58-60, July, 1998.) These programs are also available on-line at the Attotron web site (www.attotron.com).

BUGS, GLITCHES, AND BIZARRE BEHAVIOR

  1. Yes, there are spelling and grammatical errors, as well as technical inaccuracies in the Virtual Catalog (though the restriction sites should all be accurate). Yes, it is embarrassing to show misspelled documents to teachers. I promise to spell check the public release versions.

  2. The most serious "bug" is that some version of the Netscape browser crash when Cybertory runs. Please note the known compatabilities and incompatibilities at the beginning of this letter.

  3. Occasionally, for no fathomable reason, clicking on objects in the restriction digestion module will cause a file location to be magically pasted into a data element of the top frame. When this happens, press DELETE to erase the file location information, and use the scroll bar on the right side of the top frame to bring it back to the top, with the logos and the pull-down menu. You may also need to click in the space below the timer before the timer will accept a button click. We have no idea why this bug happens, but we may be able to sidestep it in future releases.

  4. Clicking on a tube label, rather than on the tube behind the label, will not select or deselect the tube. Move the cursor off of the label and on to the tube, and it should change from an arrow to a hand, indicating it is over a functioning hot spot.

  5. Sometimes you need to click things more than once.

  6. Holding the Macintosh mouse button down too long brings up a navigation menu (this is called the "context sensitive menu." It is the same menu you get if you right-click in Windows. On the Macintosh, it comes up when you click and hold. Unfortunately, the navigation menu doesn't make much sense in the context of the Virtual Laboratory.)

  7. Dragging the fluorescent ruler can be tricky on the Macintosh, because the context sensitive menu will pop up if one doesn't start dragging soon enough.

  8. The individual Analysis Tools do not have buttons to go back to the page from which you can choose another tool. For now, use the "context sensitive menu" to go back in frame (right click in the background of the lower frame in Windows, or click and hold on the Macintosh)

  9. The LED on the gel box does not work on some copies of the CD-ROM (it appears to be burned out! This was NOT intentional; there is presumably an error in the image of the red LED on some CDs.) Even if the LED does not work, you can tell that the UV light is on because there should always be marker DNA in the rightmost lane, and the ruler should only be visible in the dark if the UV light is on.

  10. Depending on the settings used for Netscape's Preferences, the information in the top window may not be positioned properly. Specifically, if the preferences are set for a font larger than about 12 points, the simulation module pull-down menu may not be visible. Simply scroll down a bit to see it. If the volume indicator on the pipet is too large, this also may be due to use of a font size greater than 12 points.

BETA TESTER AGREEMENT

A downloadable (zipped) version of this program is available to scientists and teachers who join the beta testing program. This version can be used off-line, and should be useful for classrooms without network connections. Qualified individuals agreeing to take a copy of the program should sign and return the Beta Tester Agreement. Beta Testers who use the prototype in their own classrooms may continue to use it indefinitely without charge, but they must not distribute it outside of their classes without permission from Attotron Biosensor Corporation. Our marketing plan is still being formulated, but we hope to come up with a plan that will let us distribute at least a useful core portion of the program at no cost, yet still retain the potential for commercial profitability, as required by the SBIR funding mechanism. We hope to be able to sell accessory materials, workbooks, teacher's manuals, curricula, etc., and perhaps web space advertising. Though we hope to be able to give software away, we ask that other people not give our software away without permission.

In particular, the Beta Testers agreement requires the testers not to place this program on any web server that might be accessible to the World at Large. For local area networks, it can be used from a simple file server (bookmarks can even be placed to a file on the server); a web server should not be necessary in most cases. Anyone who would like to use the prototype on a network should write to


            Robert M. Horton, Ph.D.

            Director of Research

            Attotron Biosensor Corporation

            106 East Adams Street, Suite 210

            Carson City, NV 89706 USA

or (preferably) send e-mail to rmhorton@attotron.com to discuss it.

All beta testers will be signed on to the Cybertory mailing list (send subscription requests here), a moderated list server which will be used exclusively for discussion and feedback about Cybertory and supporting software and teaching ideas. New bug reports should be submitted to the mailing list.