Plasmid‐free T7‐based Escherichia coli expression systems

Abstract

In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing.

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

“…Traditional protocols for replacing large segments of bacterial genomes with heterologous genes or genetic operons could be vastly improved by implementing the utility of CRISPR/Cas9 selection. Here we are proposing an improvement on such earlier methods (3,9,10) achieved by simplifying the protocol for chromosomal gene replacement and abolishing the reliance on chromosomal antibiotic markers and the use of Flp recombination for antibiotic marker recycling. We have shown that E. coli gene replacement can be greatly simplified through application of the CRISPR/Cas9 system.…”

“…A mixture of target modified cells and unmodified wild-type cells that escaped Cas9 cleavage are obtained via selection of pCas9 plus pCRISPR cotransformants and can be easily differentiated using PCR screening. In contrast to traditional recombineering protocols for chromosomal gene replacement (3,9,10), CRISPR/Cas9 recombineering is simpler, is less labor-intensive, and can be carried out in a substantially shorter time frame (Fig. 5).…”

“…Relying on either RecET from the Rac prophage (2) or the bacteriophage lambda Red proteins, Exo, Beta, and Gam (1), recombineering allows simple and efficient construction of gene knockout mutants via homologous recombination of a double-stranded DNA (dsDNA) PCR product with bacterial chromosomes (3). This work has led to the construction of an Escherichia coli K-12 single-gene knockout library (the Keio collection [4]), enabled widespread use of chromosome-encoded expression platforms for metabolic and strain engineering (5)(6)(7)(8)(9)(10)(11), and simplified conventional recombinant DNA technology through the advent of ligation-independent DNA cloning (2,12). Automated, high-throughput, and multiplexed genome editing applications have also been envisioned, including multiplexed automated genome engineering (MAGE), generating up to 4.3 billion genomic mutants per day (13).…”

Antibiotic-Free Selection for Bio-Production: Moving Towards a New "Gold Standard"

“…This system also conferred a high stability and simple upstream processing as well as high flexibility in process design. (Striedner et al, 2010).…”

Process analytical technology (PAT) for biopharmaceuticals

“…For example, based on these results an improved transcription tuning concept, utilizing the real-time predicted cell dry weight (CDW) for setting of a constant inducer to CDW ratio, has been established (to be published). Integration of comprehensive process data also yields an increased process understanding and new approaches for cell design [34]. In this work it was also demonstrated that the reduction of the induction level shows a significant increase of the soluble recombinant protein fraction.…”