A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors

Clustered PV inverters in LV networks: an overview of impacts and comparison of voltage control strategies

High penetration of photovoltaic (PV) inverters in low voltage (LV) distribution network challenges the voltage stability due to interaction between multiple inverters and grid. As the main objective is to provide more power injection from VSC-based PV inverters, grid stability, reliability and power quality must be maintained or improved by adding cooperative control features to the grid-connected inverters. This paper first gives an overview of bilateral impacts between multiple distributed generations (DG) and grid. Regarding of these impacts, recent advances in static grid voltage support functionalities to increase penetration level are compared considering voltage rise limitation. Steady-state simulation study is realized in PSCAD/EMTDC and the results are discussed in terms of total generation efficiency.Peer ReviewedPostprint (published version