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Abstract
We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
Highlights
Current protocols for assembling variant gene libraries have evolved from the relatively simple early protocols that generated random variability through error prone PCR [1] into a rich variety of protocols that allows introduction of virtually any type of variation in any given gene [2,3,4]
One obvious reason is that current cloning methods are usually not efficient enough to generate the large number of variants required for DNA shuffling
Two DNA shuffling strategies have earlier been developed based on the use of type IIB or type IIs restriction enzymes [17,18,19,20]
Summary
Current protocols for assembling variant gene libraries have evolved from the relatively simple early protocols that generated random variability through error prone PCR [1] into a rich variety of protocols that allows introduction of virtually any type of variation in any given gene [2,3,4]. One obvious reason is that current cloning methods are usually not efficient enough to generate the large number of variants required for DNA shuffling. Two DNA shuffling strategies have earlier been developed based on the use of type IIB or type IIs restriction enzymes [17,18,19,20]. These protocols are quite complex to perform, require several successive steps, and in many cases still rely on PCR for amplification of the library since only small amount of recombinant templates is obtained
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