Abstract
BackgroundIn vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction. However, the current in vivo assembly methods are not consistent with respect to yields of correctly assembled constructs and standardization of parts required for routine laboratory implementation has not been explored. Here, we present and evaluate an optimized and robust method for in vivo assembly of plasmids from overlapping DNA fragments in S. cerevisiae.ResultsTo minimize occurrence of misassembled plasmids and increase the versatility of the assembly platform, two main improvements were introduced; i) the essential elements of the vector backbone (yeast episome and selection marker) were disconnected and ii) standardized 60 bp synthetic recombination sequences non-homologous with the yeast genome were introduced at each flank of the assembly fragments. These modifications led to a 100 fold decrease in false positive transformants originating from the backbone as compared to previous methods. Implementation of the 60 bp synthetic recombination sequences enabled high flexibility in the design of complex expression constructs and allowed for fast and easy construction of all assembly fragments by PCR. The functionality of the method was demonstrated by the assembly of a 21 kb plasmid out of nine overlapping fragments carrying six glycolytic genes with a correct assembly yield of 95%. The assembled plasmid was shown to be a high fidelity replica of the in silico design and all glycolytic genes carried by the plasmid were proven to be functional.ConclusionThe presented method delivers a substantial improvement for assembly of multi-fragment expression vectors in S. cerevisiae. Not only does it improve the efficiency of in vivo assembly, but it also offers a versatile platform for easy and rapid design and assembly of synthetic constructs. The presented method is therefore ideally suited for the construction of complex pathways and for high throughput strain construction programs for metabolic engineering purposes. In addition its robustness and ease of use facilitate the construction of any plasmid carrying two or more genes.
Highlights
In vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction
To estimate the frequency of false-positive transformants resulting from utilization of linearized plasmid backbones, we took plasmid backbones used in two recently reported S. cerevisiae based in vivo assembly methods [17,18]
The BamHI fragment was 2.5 fold larger than the SspI fragment, leaving much more chance for circularization by internal recombination on short homologous sequences. These results showed that use of backbones obtained by restriction from standard yeast vectors are a serious factor in determining the fidelity of the system
Summary
In vivo recombination of overlapping DNA fragments for assembly of large DNA constructs in the yeast Saccharomyces cerevisiae holds great potential for pathway engineering on a small laboratory scale as well as for automated high-throughput strain construction. Several in vitro techniques have been developed to deal with these issues Methods such as SLIC [3], InFusionTM [4], and Gibson’s isothermal assembly [5], enable efficient assembly of up to six overlapping DNA fragments into a plasmid. In vivo assembly of multiple overlapping DNA fragments by homologous recombination in Saccharomyces cerevisiae does not exhibit these limitations [6,7,8,9] In this method, yeast is transformed with a mixture of multiple linear DNA fragments, which assemble through homologous recombination of overlapping terminal sequences [10]. Pioneering work in the 1980s already made use of this method to assemble circular plasmids [11], its application remained limited, probably due to the difficulties in the generation of the terminal homologous sequences required for recombination of the linear fragments. In vivo assembly ( known as transformation associated recombination (TAR)) was used for the cloning of large DNA fragments that were resisting traditional methods based on restriction-ligation [12]
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