Abstract
Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology.
Highlights
Recent progress in synthetic biology has made it possible to engineer gene regulatory networks with predictable behaviors [1]
We made a collection of promoter and open reading frame (ORF) Entry clones by Gateway BP recombination reactions (Table 2)
The constructed Destination vectors (Fig. 1) can be recombined with a promoter Entry clone and an open reading frame (ORF) Entry clone in a one-step Gateway LR reaction in vitro (Fig. 2)
Summary
Recent progress in synthetic biology has made it possible to engineer gene regulatory networks with predictable behaviors [1]. Construction of de novo gene networks requires efficient assembly of DNA sequences including promoters and ORFs encoding proteins with defined functions. A recent study applied this cloning technology successfully to the yeast one-hybrid system [3] In this system a library of DNA bait sequences and ORFs encoding the reporters (His and b-galactosidase) were assembled in a one-step Gateway recombination reaction. The Gateway vectors for the onehybrid system were not designed as versatile plasmid vectors for more general use to build artificial genes and introduce them into yeast Such vectors should facilitate the rapid characterization of new promoter-ORF combinations prior to the synthesis of an artificial genetic network in yeast
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