Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries.

Maximilian Otto,Christos Skrekas,Johan Gustafsson,Michael Gossing,Verena Siewers,Florian David

doi:10.1021/acssynbio.1c00408

Maximilian Otto, Christos Skrekas + Show 4 more

Open Access

https://doi.org/10.1021/acssynbio.1c00408

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Abstract

Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.

Highlights

The recent developments in the fields of synthetic biology and metabolic engineering have enabled us to genetically manipulate microorganisms in order to produce industrially relevant chemical compounds in a more sustainable way
The overall idea is to standardise basic genetic elements such as promoters, coding sequences, terminators etc. and collect them as a library of parts. These parts can be assembled to form devices or possibly even whole synthetic biological systems in the future.[5−7] Examples for assembly standards that have been adapted for multiple organisms, including S. cerevisiae, are GoldenBraid and Modular Cloning (MoClo).[8−13,74,75] Both approaches are based on Golden Gate assembly reactions,[14,15] using type IIS restriction enzymes, which have spatially distinct recognition and cleavage sites.[16]
Level-1 plasmids are assembled from level-0 part plasmids and, in the yeast MoClo toolkit, contain a yeast and Escherichia coli compatible plasmid backbone with a single yeast expression cassette.[8]

Summary

Introduction

The recent developments in the fields of synthetic biology and metabolic engineering have enabled us to genetically manipulate microorganisms in order to produce industrially relevant chemical compounds in a more sustainable way. This can be achieved by both, manipulating the endogenous metabolism of the cell and/or by introducing novel genes. For the assembly of level-2 plasmids dedicated assembly connector parts are used

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