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

Murray MacPherson, Yasushi Saka

Research output: Contribution to journalArticle

9 Citations (Scopus)
7 Downloads (Pure)

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 languageEnglish
Pages (from-to)130–138
Number of pages9
JournalACS Synthetic Biology
Volume6
Issue number1
Early online date16 Aug 2016
DOIs
Publication statusPublished - 20 Jan 2017

Keywords

  • gene assembly
  • yeast
  • saccharomyces cerevisiae
  • transcriptional terminator
  • bacterial artificial chromosome

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    Iain Fraser Cytometry Centre

    Andrea Holme (Manager), Linda Duncan (Senior Application Scientist), Ailsa Laird (Technician) & Kate Burgoyne (Technician)

    Institute of Medical Sciences

    Research Facilities: Facility

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