Tutorial: Chronography – Tracking Cloning History

One of the most useful features of GCK is called chronography . This feature allows you to keep track of construct history as well as to store different views of the same sequence in a single file. It is important to realize that although you will be seeing different graphics on screen, there is only a single sequence in each construct file. Chronography allows you the flexibility of displaying the same sequence in a number of ways to bring out key features.

  1. Start GCK and open the file called pBR322 in the tutorial files folder. You will see Figure 2.29. This is a fairly standard view of a construct with a few restriction sites marked. Note that the SalI site shows both the name and position for the marker. This can be set in the Format > Site Markers menu. The bottom right corner of the window shows a scale marker indicating the scale of the drawing. The scale marker can be shown or hidden by using the Construct > Display > Show/Hide Sites.
    Figure 2.29: pBR322 as Graphic
  2. Choose Construct > Display > Display Sequence to bring up Figure 2.30. As you will see, chronography can be used to store the appearance of a construct sequence as well as the graphical construct appearance. This view of the sequence is a simple one, consisting just of the sequence grouped in tens and a few sites marked.
    Figure 2.30: pBR322 as Text
  3. Choose Construct > Display > Display Graphics to return to the graphical view as in Figure 2.29. All the chronography features are accessible in the Format > Chronography submenu. Each different view of the construct is referred to as a different generation. Notice that at the bottom of the Chronography menu are three different named generations – restrictions sites, regions of interest, and DNA sources. These are the three different generations present in the pBR322 construct. The generation currently being shown is checked in the menu. Choose Format > Chronography > Show Previous Generation. This will bring up Figure 2.31, which is one generation in the past and is referred to as generation#1 (the most recent generation is referred to as generation#0). In this generation the coding regions are shown along with an origin of replication.
    Figure 2.31: pBR322 Generation#1
  4. Choose Construct > Display > Display Sequence. You will see Figure 2.33 Note that the sequence is now grouped in threes and the translation of the tetracycline resistance gene is shown. This is not a different sequence – it is just being displayed differently.
  5. Choose Construct > Display > Display Graphics and then choose Format > Chronography > Show Previous Generation again to see the most ancient generation for this construct. This is Figure 2.32. This generation shows the three different segments that were used to construct the original pBR322 plasmid.
    Figure 2.32: pBR322 Generation#2
    Figure 2.33: pBR322 Generation#1 Sequence
  6. Double-click on the blue segment from about 12 o’clock to 5 o’clock to select that segment. Now choose Construct > Get Info… [or press command-I (Mac) /ctrl-I (Windows)]. You will see Figure 2.34. This provides information about the source of each DNA segment in the construct. By carefully documenting the sources of each DNA segment, the entire history of a construct can be maintained easily. All the comments entered can also be searched using the File Searching capabilities of GCK (see Tutorial 8: Finding Comments and File Searching). Press the Cancel button to return to the graphical window. Select the entire DNA of the construct by choosing Edit > Select All [or pressing command-A (Mac)/ ctrl-A (Windows)].
    Figure 2.34: Segment Info
  7. Choose Format > Chronography > Restriction Sites to bring you back to the most recent generation of the construct. Remember, this is really just a different view of the same sequence you have been looking at throughout this tutorial.
  8. Choose File > Open and open the file called actin DNA , which is in the tutorial files folder. Double-click on the SalI fragment to select it (Figure 2.35), and then choose Edit > Copy. We will be cloning this fragment into pBR322.
    Figure 2.35: SalI Fragment of Actin DNA
  9. Click once in the pBR322 window to make it the active window and then put the insertion point at the SalI site on the pBR322 DNA. You can do this either by clicking in the DNA at the location of the SalI marker or by clicking on the SalI marker itself. Once the insertion point is correctly located, choose Edit > Paste to paste the actin DNA SalI fragment into the pBR322 at the SalI site. You will get a warning that you are about to delete a region (the tetracycline region in generation#1 is there, even though you cannot see it – click OK) and then you will see Figure 2.36. Note that the positions in the SalI markers have been updated and the size of the construct has been updated in the center of the window.
    Figure 2.36: Actin Fragment into pBR322
  10. Choose Edit > Select All to select the entire construct and then choose Format > Chronography > Show Previous Generation . This will bring up Figure 2.37. What you see here is that the previous generation of the actin DNA was copied when the actin fragment was copied. All generations of a fragment of DNA are copied when that fragment is copied. This provides an easy way to keep track of where all the different pieces of DNA have come from when you assemble a construct of your own. If comments are entered with each segment, it is always possible to figure out the entire construct history by looking at different generations.
    Figure 2.37: Actin Fragment into pBR322, Generation#1
  11. Again, choose Format > Chronography > Show Previous Generation again. This brings up Figure 2.38. Notice here that the actin fragment is just a plain gray line because there was no generation specified for the actin DNA that far into the past. Click once in the DNA at the beginning of the actin fragment to deselect the entire construct.
    Figure 2.38: Actin Fragment into pBR322, Generation#1
  12. Choose Construct > Display > Display Sequence to view the construct as a sequence in generation#2. Notice how the gray from the actin fragment is also used to display the sequence itself. You can use different generations to keep different sequence appearances just as you can keep different graphic appearances.
  13. You may find that in some complex construction projects, you might accumulate a large number of generations and it might be difficult to navigate from one generation to the next by only one generation at a time. In GCK, you can jump to any generation by using the Format > Chronography > submenu. There will be a list of all generations at the bottom of this menu. Select the Regions of Interest generation to go to that generation.
  14. Explore the different formats for this generation. Change back to the graphical view now ( Construct > Display > Display Graphics ). Try selecting a segment and then changing the generation for that segment. Note how it is possible to show different generations for different segments at the same time. This provides a great deal of flexibility for displaying sequences and graphics in exactly the way you want.

This concludes the tutorial. You will not need the construct you have just made so close any open files and do not save any changes .

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2 Comments

  1. Eric Carstens
    Posted October 11, 2012 at 6:23 am | Permalink

    I tried phoning but the line is busy. I have never used chronography but thought I would give it a try. I have a number of mutations in a construct that I want to track. However, when I went to start a new generation, the “keep displayed comments” box was dimmed. I would like to maintain this information in the next generation so I can easily review the comments about the mutated nt at that position. Is this unreasonable? Is there some way to keep the comments?
    Thanks
    Eric

  2. Posted October 11, 2012 at 8:02 am | Permalink

    Hi Dr. Carstens –

    Thanks for the note … the ‘Keep Displayed Comments’ box will be dimmed out if you currently have the construct display set to “Hide Comments” (the same holds true for Regions and Site Markers … if they are set to ‘Hide’, then those check boxes will be grayed out in the generation dialog).

    You can fix this by choosing the ‘Construct > Display > Show Comments / Show Regions / Show Markers’ menu option (to toggle them on). This should ‘turn on’ the comment indicator lines in the map, and when you choose to make a new generation, the check box should no longer be grayed out.

    Hope this helps!
    Brant

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