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Designing a Gateway Cloning Strategy

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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Cloning


Branch: Protocol-Wiki
Type: Protocol
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
31.01.2012 - Christopher Dyer  -  Sci-Mate   
13.12.2010 - John Stewart   


To place a sequence of DNA into and appropriate vector, in the correct frame and orientation, a cloning strategy is required. As per traditional cloning, a series of sequence models for all constructs is the surest approach. This protocol assumes understanding of basic cloning strategies, and focuses on Gateway specific elements of PCR insert design and vector selection.

Contents

[edit] Identifying the Sequence and Necessary Vectors

The sequence is of course, the purpose of the cloning reaction. It is generally a gene of interest, which needs to be put into the necessary vector for expression or sequencing, etc.

The necessary vector is generally an expression vector, bacterial or mammalian, or other vector containing critical features of interest. There may also be a sub-cloning step involved, which will be identified in the Cloning Strategy below.

The Gateway system relies on all sequences and vectors having the Gateway Lambda Phage Restriction Sites. If they do not have the required sequences, then this must be delt with in the Cloning Strategy. Basically, the quicker you can include the Gateway sites around sequence or within vectors, the easier and quicker the cloning can be done (see Designing Gateway PCR Primers and Creating a Gateway Vector).

There are many commercial Gateway clones available within the library of clones listed on the suppliers of the Gateway technology. Some institutions have access to Gateway libraries through their core services facilities, or colleagues may be prepared to share such clones.

The choice of a "Donor Vector", for a possible sub-cloning step, is not so as important as the choice of "Destination Vector" (where you want the sequence to end up). To choose either, go to the supplier's site and look for Donor or Destination vectors (indicated by pDONR or pDEST) with the required features. These are often fairly easy to find if your institute is aware of this technology.

If Gateway clones and vectors can be obtained, this can simplify the Cloning Strategy to a series of basic Gateway cloning reactions, see below.

[edit] Cloning Strategy

All Gateway cloning strategies are based upon the following reversable reactions (see Gateway Cloning):

B1-gene-B2              +   P1-ccdB-P2             <=>       L1-gene-L2       +         R1-ccdB-R2

In terms of inserts and vectors, the above reactions corresponds to the following recombinations:

G-W PCR Product  +   Donor vector            <=>       Entry vector                +         junk fragment

Expression vector   +   Donor vector            <=>       Entry vector                +         Destination vector

In terms of antibiotic resistance, and other selection mechanisms:

Variable                 +  Kanamyacin & ccdB  <=>       Kanamyacin         +        variable & ccdB

Basic Gateway cloning involves moving genes back and forth between "Expression vectors" and "Entry vectors".

To bring a sequence or gene into the Gateway system, see Bringing a Sequence or Gene into the Gateway System below.

[edit] Moving Sequences Within the Gateway System

If the desired sequence is already flanked by either B1-gene-B2 or L1-gene-L2 sites, and the intended vector contains either the P1-ccdB-P2 or R1-ccdB-R2 sites, then the cloning strategy is as simple as one or two basic Gateway cloning reactions.

Because all Gateway vectors are in-frame and oriented the same way as each other, very little strategy is required if the gene has been sequenced or demonstrated to express from within a Gateway vector. It can be assumed, in this case, that one can move the gene into any other Gateway vector and it will remain in frame and in the right orientation.

Of course, if N- or C-terminal tags are being introduced, it advisable to check if these elements are in-frame (not all genes were inserted with this in mind). 

[edit] Bringing a Sequence or Gene Into the Gateway System

If your sequence or gene of interest is not flanked by the needed restriction sites, then B1 and B2 sites can be added during PCR to create a G-W PCR Product (see Designing Gateway PCR Primers).

An alternative to adding Gateway elements to a PCR product is to use the TOPO system, which is prominant on the supplier's site. The appeal of this system is that only one cloning reaction is required, not two with a G-W PCR Product strategy, and it is not necessary to add 31 bp to the PCR primers. However, Gateway cloning reactions are easy and very reliable. Also, it is absolutely no problem to add 31 bps to a primer. Even if the primer is already too long, two sets of primers can be used to amplify the required additional bases. This author considers TOPO to be an even more expensive (and unnecessary) system to buy into with its own enzymes and vectors, most of which are highly specific and not as flexible or reliable as the Gateway system.

[edit] Creating Fusion Constructs, Mutations or Other Post-Ligation Modifications

The Gateway system cannot directly assist with the creation of fusion constructs, mutations, or other sequence modification techniques. However, it can still save time if an initial construct must be cloned, or the final product must be moved between vectors. Moreover, during the PCR, while adding the Gateway elements, bases can be added to facilitate traditional cloning strategies, or mutations in the original sequence, etc.

[edit] Creating a Gateway Vector

If the desired vector does not have these sites, then a R1-ccdB-R2 cassette can be inserted at an appropriate site to create a Gateway Destination vector.

[edit] Creating Sequence Models

The surest way to know where you are going, and how to get there is still to create a sequence model of the intended DNA fragment inserted into the Destination vector. With a sequence model, one can ensure that the final sequence is in-frame, correctly orientated, and contains enough promotor and start/stop elements to ensure expression of the desired product. From this point, one can work backwards to ensure that the cloning strategy delivers the right product in the end.

The following instructions to create sequence models assume that sequence is available for the intended Destination or Donor vectors; and the gene or sequence of interest is already flanked by Gateway restriction sites (as either PCR product or within any Gateway vector). If the sequence for a Gateway PCR product is not already available, it can be created by Designing_Gateway_PCR_Primers.

[edit] Creating a Gateway Expression Vector Sequence Model

Assuming you already know the basic sequence of your final sequence follow these steps to move the sequence from the source (PCR product, Expression vector, or Entry vector) into the destination vector:

  1. From the intended destination vector (pDEST...), first remove (delete) all bases starting from and ending with the underlined bold region (including the bold underlined bases): 5'-ACA-AGT-TTG-TAC-AAA-AAA-GCT-GAA-C-ccdB gene-GTT-CAG-CTT-TCT-TGT-ACA-AAG-TGG-T-3';
  2. Insert into the destination vector (at exactly the same place where the bases were deleted), sequence from the souce vector starting from and ending with the underlined bold region (including the bold underlined bases): 5'-(A/C)CA-A(C/G)T-TTG-TAC-AAA-AAA-GCA-GGC-T-sequence_or_gene_of_interest-A-CCC-AGC-TTT-CTT-GTA-CAA-AGT-(A/G)G(T/C)-3'.

The final recombined sequence should create the B1-gene-B2, characteristic of an Expression vector, see Gateway_Cloning and Gateway Lambda Phage Restriction Sites.

[edit] Creating a Gateway Entry Vector Sequence Model

If you need to create a sequence model for an intermediary Entry vector, follow these basic steps:

  1. From the relevant Donor vector (pDONR), delete all bases starting from and ending with the underlined bold region (including bold underlined bases): 5'-CCA-ACT-TTG-TAC-AAA-AAA-GCT-GAA-C-ccdB gene-G-TTC-AGC-TTT-CTT-GTA-CAA-AGT-TGG-3' (including all bases in between, upto and including the bases underlined in bold);
  2. From either the PCR product, Expression vector or Entry vector, insert all bases starting from and ending with the underlined bold region (including the bold underlined bases): 5'-(A/C)CA-A(C/G)T-TTG-TAC-AAA-AAA-GCA-GGC-T-sequence_or_gene_of_interest-ACC-CAG-CTT-T-CTT-GTA-CAA-AGT-(A/G)G(T/C)-3'.

The final recombined sequence should create the L1-gene-L2, characteristic of an Entry vector, see Gateway_Cloning and Gateway Lambda Phage Restriction Sites.

[edit] Analysis

Perform a thorough analysis of the sequence to be sure that it contains all the necessary elements; that they express themselves as ORFs; and that no other unwanted elements have been introduced. If the strategy involves creating a Gateway PCR product, then the sequence should also be compared to the sequence designed when designing Gateway PCR primers.

If mistakes are recognized or problems exist, revise the strategy to correct the issue(s).

[edit]





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