Simple, Efficient and Versatile - Cloning by homologous recombination in Saccharomyces cerevisiae

by Steven Buckingham Labtimes 05/2014




Homologous recombination (HR) of ‘meiotic’ noodles. HR is also at the heart of the recently introduced AGAP-cloning system. Image: Hunter Lab /UCLA Davis

Cloning by homologous recombination in Saccharomyces cerevisiae is fairly easy but not widely adopted because it is incompatible with most plasmids used in standard cloning procedures. But not anymore.

Modern molecular genetics experiments need a set of sophisticated tools for manipulating genetic material. These tools need to be quick, cheap and versatile. The call for versatility means that such tools have to be easy to insert into just about any workflow, without having to go through tedious optimisation steps each time you turn them to a new use. The call for cheapness means we don’t want to have to buy expensive kits. And quick means: I want to be able to do several steps at once and do them in as short a time as is reasonable.

As if to defy this wish-list, practical cloning can often turn out to be expensive, inflexible and slow. For sure, there is a battery of novel and clever ways to adapt cloning strategies, so that they can be plugged into just about any workflow. But each one of them, whether it be the Gateway or the TOPO-TA systems, or indeed just about any of the lab-generated techniques, suffer from drawbacks, such as cost of reagents or the need to optimise afresh each time you modify an experiment.

On the other hand, cloning using the yeast homologous repair method comes just short of being the perfect answer to all our cloning woes. It is efficient and very robust, and all you need to get it working for the DNA you want to clone is to tack on 29 nucleotides identical to a region in the host plasmid. The problem is, you need to have a yeast-compatible shuttle vector. So, forget about cloning your chimp gene with this. Argh!

Simple idea

But this limitation is overcome in a remarkably simple adaptation to the yeast recombination cloning method announced earlier this year (Joska et al., J Microbiol Methods 100: 46-51). The protocol is called any-gene-any-plasmid-cloning, or AGAP-cloning, and the idea is very simple. You take a plasmid of your choice and insert not only your own sequence (adapted for repair cloning) but also a yeast cloning cassette (YCC). The YCC contains code that allows expression to be driven in yeast as well as selection and it frees you from having to use a yeast-compatible vector.

So, how is it done in practice? First, you select the plasmid you want to work with − it can be any plasmid you want, it doesn’t have to be a yeast or E. coli one. The plasmid is then cut at two or more sites using a couple of restriction enzymes. These sites can both be in the Multiple Cloning Site if you prefer, or one of them can be elsewhere, provided of course the cutting does not interfere with any essential sequence such as a selection sequence or a promoter. This ­produces two linearised fragments.

Next, you synthesise the DNA strand you want to clone and tack on to the 5’-end a stretch of 29 nucleotides identical to the enzyme-cut site. Other than that, there is no other restriction to the DNA sequence.

Widespread use

Last of all, you need the Yeast Cloning Cassette. This consists of two parts: the yeast 2-micron origin of replication (2 µm ori) that directs transcription in yeast and the URA3 gene that allows you to select the correctly transformed cells.

The mixture of the two linearised strands, along with the adapted DNA and the Yeast Cloning Cassette, is then transformed into a yeast strain lacking a functional URA3 gene. Cells selected by the Ura3 selection pressure are then harvested and the construct recovered by transforming into E. coli. Job done!

The method has several features that commend it to widespread laboratory use. First, there is no limitation as to the plasmid used, or the gene to be cloned and no ligation steps are involved. Second, it can be used to clone several DNA sequences simultaneously by judiciously concatenating the sequences together and tagging on the 29 identical residues.

The method is also very versatile. For one, you aren’t stuck with yeast or E. coli. The authors of the article report that they successfully applied the technique outside the E. coli/S. cerevisiae combination by transforming Staphylococcus aureus with a two-component regulatory system that works as an operon. Even better than that, the method isn’t even constrained to prokaryotes: the authors also used the method to tag a zebrafish gene with a green fluorescent protein marker, something that otherwise cannot be done without ligase-based cloning. Finally, they showed that you can use the method to conveniently introduce site-directed mutations, simply by using synthesised, 29-nucleotide-tagged DNA with the altered sequence.

Fast and cheap

The method is claimed not only to be cheaper but faster than its competitors. For one, it means that provided the PCR yields a single band, there is no need to ­gel-purify PCR fragments. On top of that, according to the authors, the preparation of the plasmids used up a mere six hours or so of bench time.





Last Changed: 16.09.2014




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