Abstract
Assembly of synthetic genetic circuits is central to synthetic biology. Yeast S. cerevisiae in particular has proven to be an ideal chassis for synthetic genome assemblies by exploiting its efficient homologous recombination. However, this property of efficient homologous recombination poses a problem for multigene assemblies in yeast since repeated usage of standard parts, such as transcriptional terminators, can lead to rearrangements of the repeats in assembled DNA constructs in vivo. To address this issue in developing a library of orthogonal genetic components for yeast, we designed a set of short synthetic terminators based on a consensus sequence with random linkers to avoid repetitive sequences. We constructed a series of expression vectors with these synthetic terminators for efficient assembly of synthetic genes using Gateway recombination reactions. We also constructed two BAC (bacterial artificial chromosome) vectors for assembling multiple transcription units with the synthetic terminators in vitro and their integration in the yeast genome. The tandem array of synthetic genes integrated in the genome by this method is highly stable because there are little homologous segments in the synthetic constructs. Using this system of assembly and genomic integration of transcription units, we tested the synthetic terminators and their influence on the proximal transcription units. Although all the synthetic terminators have the common consensus with the identical length, they showed different activities and impacts on the neighboring transcription units.
Original language | English |
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Pages (from-to) | 130–138 |
Number of pages | 9 |
Journal | ACS Synthetic Biology |
Volume | 6 |
Issue number | 1 |
Early online date | 16 Aug 2016 |
DOIs | |
Publication status | Published - 20 Jan 2017 |
Bibliographical note
The authors thank the assistance of the Iain Fraser Cytometry Centre at the University of Aberdeen. We also thank Claire MacGregor, Diane Massie and Yvonne Turnbull for technical assistance, Alexander Lorenz and Ryohei Sekido for critical reading of the manuscript and Richard Newton for preliminary results. This work was supported by Scottish Universities Life Sciences Alliance (SULSA).Keywords
- gene assembly
- yeast
- saccharomyces cerevisiae
- transcriptional terminator
- bacterial artificial chromosome
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Iain Fraser Cytometry Centre
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