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).Peer reviewedPostprin

MacPherson, Murray

Saka, Yasushi

ACS Synthetic Biology

English

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 few 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

Murray MacPherson (583035)

Yasushi Saka (83561)

The Francis Crick Institute

Short Synthetic Terminators for Assembly of Transcription
Units in Vitro and Stable Chromosomal Integration in Yeast S. cerevisiae

Murray MacPherson

Yasushi Saka

Crossref

Short Synthetic Terminators for Assembly of Transcription Units in Vitro and Stable Chromosomal Integration in Yeast S. cerevisiae

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).Peer reviewe

Aberdeen University Research

Short Synthetic Terminators for Assembly of Transcription Units in vitro and Stable Chromosomal Integration in Yeast S. cerevisiae

https://aura.abdn.ac.uk/bitstream/2164/9159/1/acssynbio_final_ver.pdf

Short Synthetic Terminators for Assembly of Transcription Units in vitro and Stable Chromosomal Integration in Yeast S. cerevisiae

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The Francis Crick Institute

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Aberdeen University Research