Molecule Construction and Design Using Vector NTI 10 AdvanceYi-Bu Chen, Ph.D.Bioinformatics SpecialistNorris Medical Library University of Southern California323-442-3309 yibuchenbelen.hsc.usc.eduWorkshop OutlineA.Overview of molecule types and creation methods B.Major steps and tools for molecule construction and designC.Construct a new DNA moleculeD.Design a new DNA molecule E.Overview of Gateway and TOPO cloningF.Using simulated gel electrophoresis to analyze cloned productsG.Useful online resources for molecule design purposeFundamental Molecule Types in Vector NTIv Basic Molecules (DNA/RNA/Protein) Not built from component fragments Sequences and features are either entered by users or imported from other databases. v Constructed DNA/RNA Molecules Built from one or more fragments (parent molecules, linkers, adapters, etc.) Automatically receive the Feature map and sequences from the parent molecules.v Constructed Protein Molecules Translated from a coding sequence of a DNA molecule Does not receive the Feature map from its parent DNA moleculeMethods of Creating New Molecule in Vector NTIv Basic Molecules (DNA/RNA/Protein) Importing molecules or sequences Manually creating new molecules v Constructed DNA/RNA Molecules Splicing exons of an intron-exon join feature of another molecules Constructing from compatible component fragments from other molecules Designing from components of a user-defined fragments list Back-translating from protein molecules from components of a user-defined fragments listv Constructed Protein Molecules Splicing exons of a gene Translated from DNA moleculesMolecule Construction vs. Molecule DesignvRecipient and donor fragments are defined by users.vRestriction sites are defined by users.vWhen required, the methods of terminus modification are defined by users.vRecipient and donor fragments are selected by users.vRestriction sites are analyzed and generated by Vector NTI.vMethods of terminus modification are determined by Vector NTI.Major Tools for Molecular Construction and Designv Fragment Wizard Construct fragments (define positions and termini) Design recipient/donor fragments (define termini) Add defined fragments to the Goal Molecule Definition List v The Goal Molecule Definition List Contains the list of fragments defined and added by the user using the Fragment Wizard Serves as the starting point of molecular construction and design Designs from components of a user-defined fragments listv The Construct/Design Molecule Dialog Box Allows users to set the construction parameters and design preferences Allows users to enter information about the new moleculeMajor Steps for Molecule Construction1. Use Fragment Wizard to define component fragments.2. Add defined fragments to the Goal Molecule List.3. Use the Construct Molecule Dialog Box to set construction parameters, including necessary terminus modifications.4. Name, select data and describe the new molecule.5. Verify and edit the component fragments in the Goal Molecule Definition List.6. Initiate molecule construction.A Molecule Construction Examplev Task Clone a fragment from pBR322 into pUC19 Donor fragment: pBR322, 5EcoRI3AvaI Recipient fragment: pUC19, 5SmaI3EcoRI v Getting started 1. Open pBR322 and pUC19 in Vector NTI2. Arrange the display window to display 2 molecules on the same screen: Select Menu Window Tile Vertical A Molecule Construction Example Step 1: Describe component fragments in the Fragment WizardA. Define the recipient pUC fragment (5SmaI3EcoRI) 1. Activate the pUC Graphic Pane2. Click Add Fragment to Goal List button to open the Fragment Wizard3. 1st screen: select Construct fragment click Next4. 2nd screen: Click on the SmaI (415 bp) site in the graphic pane to set the 5 terminus. Click Next5. 3rd screen: hold the SHIFT key and click on the EcoRI site in the graphic pane to set the 3 terminus. Click Finish6. Check the description of the fragment in the New Fragment message box. Click Cancel to go back to the Fragment Wizard if there are errors, otherwise, click Add to List to add the 1st fragment to the Molecule Goal List.B. Define the donor pBR322 fragment (5EcoRI3AvaI) following the above steps1. From the tool bar, click the Open Goal List button 2. Notice that the 1st fragment in the list is always considered as the “recipient” molecule; the order of the fragments can be changed by click the Up or Down buttons 3. If no errors, click the Run button in the Lists screenA Molecule Construction Example Step 2: Inspect the Goal ListA Molecule Construction Example Step 3: Describing the new molecule1. In the Construct Molecule screen, enter name “Tutorial1” 2. Click the Recipients Start button to define the start of the new molecule (can be set at any other positions) 3. Click General Info button to enter more info in the General Data dialog box (Description: Tutorial molecule#1; Extra- Chr