ABSTRACT
Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production.
Subject(s)
Bacterial Proteins/genetics , CRISPR-Cas Systems , DNA, Fungal/genetics , Endonucleases/genetics , Genetic Engineering/methods , RNA, Guide, Kinetoplastida/genetics , Saccharomyces cerevisiae/genetics , Bacterial Proteins/metabolism , CRISPR-Associated Protein 9 , Clustered Regularly Interspaced Short Palindromic Repeats , DNA, Fungal/metabolism , Endonucleases/metabolism , Gene Expression , Isomerases/genetics , Isomerases/metabolism , Plasmids/chemistry , Plasmids/metabolism , Promoter Regions, Genetic , RNA, Guide, Kinetoplastida/metabolism , Saccharomyces cerevisiae/metabolism , SoftwareABSTRACT
A critical feature of state-of-the-art microfluidic technologies is the ability to fabricate multilayer structures without relying on the expensive equipment and facilities required by soft lithography-defined processes. Here, three-dimensional (3D) printed polymer molds are used to construct multilayer poly(dimethylsiloxane) (PDMS) devices by employing unique molding, bonding, alignment, and rapid assembly processes. Specifically, a novel single-layer, two-sided molding method is developed to realize two channel levels, non-planar membranes/valves, vertical interconnects (vias) between channel levels, and integrated inlet/outlet ports for fast linkages to external fluidic systems. As a demonstration, a single-layer membrane microvalve is constructed and tested by applying various gate pressures under parametric variation of source pressure, illustrating a high degree of flow rate control. In addition, multilayer structures are fabricated through an intralayer bonding procedure that uses custom 3D-printed stamps to selectively apply uncured liquid PDMS adhesive only to bonding interfaces without clogging fluidic channels. Using integrated alignment marks to accurately position both stamps and individual layers, this technique is demonstrated by rapidly assembling a six-layer microfluidic device. By combining the versatility of 3D printing while retaining the favorable mechanical and biological properties of PDMS, this work can potentially open up a new class of manufacturing techniques for multilayer microfluidic systems.
