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Vector NTI Tutorial: Chapter 9 Vector NTI Tutorial Chapter 9. Tutorial: Advanced Molecule Design Background Note This chapter assumes that you have completed the preceding tutorials and are familiar with Vector NTI’s user interface, selection of molecule fragments, the Goal Molecule Definition List and molecule design with the aid of Vector NTI’s built-in biological knowledge. This chapter introduces you to more complex Vector NTI cloning situations. Now that you have completed a simple molecule design, let’s look at two more complicated cases. First we will assign some complex conditions to the recipient molecule while leaving the donor relatively simple; then, we will use a relatively simple recipient but make the donor more complex. Since you are probably getting tired of pBR322 and pUC19, let’s change molecules.
We will now use BPV1 and SV40, and our overall task will be to clone the large T-cell antigen gene (LARGET) from SV40 into BPV1. Your Task: Complex cloning Your task will be to clone the LARGET gene from SV40 into BPV1 in two different ways. You will place requirements first upon the recipient and then upon the donor, and consider Vector NTI’s proposed design plans. Follow the steps in the order shown.
Figures show what your screen should look like at various points along the way. Launch Vector NTI Launch Vector NTI by double-clicking its icon in the program group or folder in which you installed Vector NTI. Open and Arrange Display windows for BPV1 and SV40 Open Molecule Display windows containing the molecules BPV1 and SV40. Use the default Display settings. You will be working with the molecules’ graphical maps, so arrange the Display windows conveniently. First Design: Complicated Recipient In the first complex design, you will insert SV40’s LARGET gene into the second ApaLI site of BPV1.
You will also require that Vector NTI save the 5’ ApaLI site and prohibit blunt–blunt fragments. If the donor has ApaLI sites appropriate for cutting the cloned fragment, then the problem will be simple. If not, the system will have to take a more complicated approach to perform the insertion. Define the recipient Switch to the BPV1 Display window and click somewhere in its graphics pane to activate it. Call up the Fragment Wizard, select the Design Recipient option on the first screen and press the Next button. To define 5’ terminus, click on the label of the ApaLI restriction site #2 in the graphics pane (nucleotide 7631, or about the 11 o’clock position).
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Click the Next button to continue. The next screen allows you to specify the save/lose condition for ApaLI site on 5’ end of the recipient. Select Save Site and press the Next button to go to the 3’ terminus screen. Now hold down the SHIFT key and click on the same site again.
The name and the position of the site are displayed in the Fragment Wizard. Press the Finish button to complete the definition of the recipient fragment. The New Fragment message box is displayed with the description of the selected recipient fragment. Check the description of the fragment in the message box.
Notice, that the 3’ and 5’ termini of the recipient fragment are set on the same ApaLI site. Press the Add to List button. The recipient fragment is added to the Molecule Goal list. Define the donor Switch to the SV40 Display window and click somewhere in its graphics pane to activate it. One for all brand nubian rare. Call up the Fragment Wizard, select the Design Donor option on the first screen and press the Next button. Click on the LARGET signal’s symbol or label in the graphics pane to select it and press the Finish button in the Fragment wizard.
Check the description of the donor fragment in the message box and press Add to List button. The donor fragment is added to the Molecule Goal List.
Inspect the Goal Molecule Definition List Now check the Goal Molecule Definition List. Press the Open Goal List button ( ) in the main toolbar and the Construct/Design Molecule dialog box appears.
The Component Fragments section of the dialog box contains the Goal Molecule Definition List. The list consists of the two fragments you defined. The recipient fragment must be the first in the Goal Molecule Definition List. Double-click the SV40 fragment, and the Fragment Editor dialog box opens.
Click the 'Inverted” check box to change LARGET’s direction to match the recipient’s direction and press OK. (You could leave LARGET in its original orientation if you want to; the system will design your new molecule either way. We have changed LARGET to inverted only to demonstrate how Vector NTI can clone fragments in different orientations.) 6. Enter general information for your new molecule Change the molecule’s name to TUTORIAL3. Press the General Info button. In the Description field, enter “Tutorial molecule #3”.
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Set the Replicon Type to Plasmid, and turn on the Bacteria Extra-Chromosome Replication option. Enter your name as a keyword. Press OK to return to the Construct/Design dialog box. Check the Recipient’s Start button in the radio button group just above the Component Fragments section to ask Vector NTI to make the start of the new molecule at the same place (if possible) where the recipient molecule (BPV1) starts. Prepare to design the new molecule Press the Design button in the upper right corner of the Construct/Design Molecule dialog box.
Vector NTI first asks you to name a subbase for results. Select the “Tutorial” subbase we created before and press OK to continue. Vector NTI saves the information you have entered about your new molecule, i.e. The name, replicon type, extra-chromosome replication abilities, list of fragments, etc. The Design Parameters dialog box appears. Leave all the settings at their default values and move on to the next step.
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Set the design preferences Now click on the Preferences button. The Design Preferences dialog box appears. Note that the blunt–blunt ligation box is already turned off. This is because Vector NTI remembers the changes you made in Chapter 7 of the Tutorial. The program remembers your design preferences so that you won’t have to set them every time you design a new molecule.
Below the check boxes are priority lists specifying which of the techniques are preferable. Leave these at their default values. Press the OK button to accept the Design Preferences and return to the Design Parameters dialog box. Design the new molecule With the goal molecule defined and your preferences for design techniques entered and prioritized, you are ready to command Vector NTI to design your new molecule. Press the Start Design button. Vector NTI gathers information from the database, generates some necessary data and then begins designing your goal molecule. “Thinking.” appears in an information box.
As before, an optimum cloning method is found within a few minutes and the goal molecule is constructed based on that best option. Inspect the new molecule When the design is complete Vector NTI opens a new Molecule Display window containing the molecule you have created. Inspect the graphical map and text description of your new molecule Switch to the text pane and open TUTORIAL3’s Design Description folder and the Step #1 subfolder. A description of how to create TUTORIAL3 appears.