Perhaps the most important window in GCK is the Construct Window . This is the place where you will do the majority of your cloning manipulations and can precisely define how your construct will look. In this tutorial you will learn how to modify the appearance of constructs. Tutorial 2: Marking Sites shows how to mark restriction sites and other features in your construct. Tutorial 7: Chronography – Tracking Cloning History shows how GCK can automatically keep track of construct history for you. Tutorial 4: Viewing the Construct as a Sequence discusses the sequence view of a construct.
- Start GCK.
- Open the file called Bluescript KS+ in the tutorial files folder by choosing File > Open… . This will bring up Figure 2.1. This folder should be in the GCK folder that was created when the program was installed. The buttons on the bottom let you choose to have listed different types of files in the list on the left. Since we are opening a construct file, leave the radio button set on Construct . When you open other kinds of GCK files, the buttons will be useful to you. Select the Bluescript KS+ file and press the Open button. You will see Figure 2.2.
- This construct has a number of segments , or sections, of DNA indicated in different colors and patterns. The title of the file is indicated in the title bar at the top of the window. In the lower left corner of the Construct window is an indicator of the location of the current insertion point . The insertion point is where new sequence would be placed into the current construct by typing or pasting, between nucleotides 625 and 626 in this window (you might have a different location in your file). Clicking with the mouse anyplace along the DNA will place the insertion point at the closest boundary between two segments.
- Double-click on the red segment of DNA to select it. This is similar to selections made in standard text editing programs, where double-clicking selects a word. This will highlight the red segment as shown in Figure 2.3. Note how the indicator in the bottom left corner now shows the range of nucleotides selected. The range is always shown in the clockwise orientation (or from left to right for linear constructs). You should see 792-625 selected. The origin (top of construct) is defined as position#1. Clicking on this popup menu will also allow you to show the position of the cursor as you move it over the DNA, or the size of the selected DNA segment.
- Once an object (like a DNA segment) is selected, you can modify it. Choose Format > Lines > and choose the second line down to change the width of the selected DNA segment to 2 pixels. In the Lines menu, you can set the width using the line thicknesses shown in the menu, or you can choose Format > Lines > Pick a Width… to type in your own thickness. Using the Format menu, you can change the appearance of the selected object. For now, do not change anything else but feel free to explore the Format menu on your own when you finish this tutorial.
- This vector has T3 and T7 promoters and a polycloning site that we want to indicate. The promoters are shown in green and the polycloning site is a striped blue. Let’s add directionality to the promoters. Before you can add arrowheads to any segment of DNA it must first be selected. The promoter segments are rather small and might be difficult to select at this magnification. So click the mouse on one of the green segments (between the blue and red segments) to place the insertion point near what we are interested in and then choose Construct > Magnification > Zoom In to enlarge the view of the construct. Zoom In again and then select the top green segment by double-clicking. You will see Figure 2.4. Nucleotides 626-645 will be selected.
- Choose Construct > Get Info… to see the name of the segment and any comments associated with that segment. This is the T7 promoter as shown in Figure 2.5. Every segment of DNA can have a name and comments associated with it. These comments can be used to store important information about the construct and can be searched using the Search Files capability of GCK (Tutorial 8: Finding Comments and File Searching). We will talk more about this dialog box later (Tutorial 7: Chronography – Tracking Cloning History). For now just press the Cancel button – you don’t want to make any changes.
- You should still have the T7 promoter selected as shown in Figure 2.4. If you do not, then select it again by double-clicking on the segment. First, let’s change the line thickness by choosing Format > Lines > and choose the third line down to change the width of the selected DNA segment to 3 pixels. Next add an arrowhead pointing in the clockwise direction by choosing Format > Lines > and choosing the rightward pointing arrow in the second section of the Lines menu.
- The last step is to adjust the arrowhead size. This is done by choosing Format > Lines > Size Arrowhead… . You will see Figure 2.6. Click and drag the mouse in the dialog box to resize the arrowhead to be approximately what is shown in this figure. Click OK .
- We now need to adjust the T3 promoter segment to point in the counter-clockwise direction. This can be done by following the same steps we just did for the T7 promoter. Briefly, do the following: Select the lower green segment (the T3 promoter) by double-clicking on it. If you cannot see this segment in the window, use the scroll-bars to bring it into view. Choose Format > Lines > and select the third line down to change thickness, choose Format > Lines > and select the leftward pointing arrow. This will add an arrowhead that is the same size as the last arrowhead created so you should have symmetrical arrowheads.
- Finally, we want to see the whole construct again, so choose Construct > Magnification > Fit to Window . This will produce Figure 2.7. We have now formatted the segments of this construct to meet our needs.
- [NOTE – if working with the demo version of GCK, saving is disabled. If you plan on continuing on to the next Tutorial, you can leave the file you just created, construct#1 , open. If not, you can quit now – simply close this file without saving the changes.]
- If working with a ‘Trial Version’ or the ‘Full Version’ of the application, you can save the modified construct under a new name. You might first want to create a folder on your desktop to hold the different tutorial files you will be creating in this and in subsequent tutorials. Having a single folder will make it easier to find the tutorial files you create as you need them in later tutorials. Choose File > Save As… , name the file myconstruct#1 , and save it in your own folder – please do not replace the tutorial files provided because others might want to use them later on. Make sure you know where you are saving this file. You will need it again later.
You can quit now, or continue on to Tutorial 2: “Marking Sites”. If you plan on continuing, you can leave the file you just created, construct#1 , open. If not, close this file.