TUTORIALS

Restriction Cloning

This tutorial shows you how to use the restriction cloning tool in Geneious Prime including an introduction to the cloning interface, and how to ligate a PCR product into an expression vector and confirm the CDS in-frame fusion.

TUTORIAL INSTRUCTIONS

Complete the tutorial yourself with included sequence data. Download the tutorial then install by dragging and dropping the zip file into Geneious Prime. Do not unzip the tutorial.

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Introduction

The Geneious Prime Restriction Cloning tool can ligate any combination of linear or circular nucleotide sequences, will automatically identify compatible cut sites, and perform one-step digestion and ligation with drag and drop ordering of two or more fragments. 

Input documents do not need to be predigested or annotated with restriction sites, as Geneious will identify the restriction sites present in your input documents and attempt to guess the sites your cloning strategy will use.  By default, restriction sites with recognition sites of 4 bp or less are not considered.  If you need the Restriction Cloning tool to use 4 bp cutters then contact the Geneious support team.

The Restriction Cloning tool can be found under menu Tools → Cloning, or via the Cloning button on the Toolbar. You can launch the Restriction Cloning tool with or without selecting your input sequences first.

In this tutorial we will ligate a PCR product into the multiple cloning site (MCS) of the expression vector pET26B (See Novagen). The pET26B vector MCS lies within a short coding sequence (CDS) that encodes an N-terminal PelB signal sequence for potential periplasmic localization and a C-terminal His•Tag® sequence.

The PCR product provided in this tutorial has been generated with PCR primers designed to amplify the region encoding the mature XynA enzyme (without signal peptide or stop codon) from the bacterium Dictyoglomus thermophilum.  The primers were designed with extensions to generate an N-terminal NcoI site and a C-terminal BamHI site.  The NcoI site has been positioned so that ligation will give an in-frame fusion to the pET26B-based PelB signal peptide.  The BamHI site is positioned so that ligation will generate an in-frame C-terminal fusion of XynA to the pET26B-based HIS-tag.

Part 1: Performing Restriction Cloning

A: Overview of The Restriction Cloning window

The Restriction Cloning window comprises 4 main sections.

The top section  allows you to set your vector (Backbone), define how the Candidate Enzymes are chosen, and if chosen from an Enzyme Set, define which enzyme set to use. The Choose… button next to this dropdown menu will allow you to select a subset of enzymes in your list if you only wish to specify a subset of enzymes within a list.

The panel below this, referred to as the “Construct layout” panel  shows tag representations of all sequences selected for the cloning operation.  You can use the Choose… button next to the Backbone dropdown menu or the Add Insert… button to add a new backbone or new insert sequences.

You can drag and drop tags to change their order, and click on the tag-associated triangle to see a dropdown menu that allows you to switch sites, reverse complement the sequence or modify digestion overhangs on each tag.

Any tag selected in panel  will be displayed graphically in the “Detailed view” panel .  The region of the sequence that will be involved in the cloning operation will be highlighted with blue shading.

The bottom section  provides options for how the Restriction cloning operations results are output.

You can click on the Help button in the bottom left corner of the window for more information on all of these options

B: Performing Restriction Cloning

The following steps describe how to perform restriction cloning using the example sequence documents provided with this tutorial.

1.  Select the pET26B vector and xynA PCR Product files that are provided with this tutorial.

2.  Go to Cloning → Restriction Cloning to open the Restriction Cloning setup window.  pET26B should be selected as the backbone and shaded blue in the Construct Layout panel, and you should see “Use Leftmost” in the Backbone dropdown menu. You can also choose a specific document from this menu if it is not set correctly.

3.  Set the Enzyme set to the Commonly used enzymes list.  In order to clone the insert into pET26B in the correct frame, we will need to use the NcoI and BamHI sites, but Geneious has automatically detected the BglII and SacI sites for cloning. BglII and BamHI have compatible cohesive ends, but the SacI site will not result in the desired ligation product. If you examine the regions and sites selected by Geneious for the cloning operation, you will see a short (210 bp) BglII-SacI fragment being selected for the backbone.

In order to select the correct BamHI and NcoI sites needed for this reaction, you will need to select a subset of enzymes from the commonly used enzymes list. Select Choose… next to the commonly used enzymes dropdown box.

This will bring up a table containing all the enzymes within the enzyme list that you have selected (commonly used enzymes). Select the tick box next to the Name column to unselect all of the currently selected enzymes, then select the BamHI and NcoI enzymes on this list. Select OK to select these enzymes and close the window.

The Restriction Cloning tool should then correctly identify and select the NcoI and BamHI sites intended for this cloning operation.

4.  If you are satisfied the settings will correctly simulate your cloning operation, then go OK.  A new file called pET26B – xynA_PCR_Product will be created.

Note: An alternative way to select other restriction sites is to click the down arrow on each document tab in the layout window, then select Choose enzyme cut site… This will bring up a menu of the available enzymes that can be used on your sequence.

Part 2: Confirming the CDS in-frame fusion

As mentioned in the introduction to this tutorial, the input PCR product for this demonstration was designed to generate an N-terminal and C-Terminal in-frame fusion between the mature xynA CDS and the CDS that spans the MCS of pET26B.

To check that the in-frame fusions have occurred correctly:

1.  Select the pET26B-XynA_PCR_Product document created by the cloning operation. The Concatenated Sequence annotations (purple) show you where the PCR product was inserted into the vector (you may need to turn these on in the Annotation and Tracks tab to the right of the viewer).

2.  Locate and select the CDS (yellow) annotation called MCS CDS PelB-MCS-His-tag.  If required, click on the Annotation tab and select it from the Annotation list.  This should select two intervals generated due to insertion of xynA. . Option (ALT)-click on the annotation to zoom in on this region.

3.  Go menu Edit -> Go to Base, then click Go to select the CDS intervals and the intervening sequence (5,072 to 6,175).

Click on the Add Annotation button, and add a new annotation called xynA fusion, of type CDS and go OK.  Note that you will get a warning that the document has an Actively linked parent. Continue with the editing.

4. A new CDS will appear.  Hover over the new CDS to see the automatic translation and confirm the CDS translates correctly over the entire CDS range. Save the document, and choose to save a copy.

5. Select and delete the preexisting CDS’s and delete them so only the XynA fusion CDS remains.  Turn on Translation in the General tab, select the Annotations tab and turn on display of Ligation annotations, then zoom in to view the translation at the fusion sites and confirm the gene product is correctly translated through the ligation sites.