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PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any position within a target plasmid. The entire process is conducted in a time-efficient and cost-effective manner, without the need of DNA gel purification and enzymatic restriction and ligation. PTO-QuickStep further innovates protein engineering by providing the possibility of integrating a random mutagenesis step (e.g., error-prone PCR) into the workflow, without compromising the time duration required. Random mutagenesis libraries can be quickly and efficiently cloned into a plasmid of interest, thereby accelerating directed evolution. On top of that, PTO-QuickStep can be utilized for rapid integration of noncoding DNA fragments to modify existing plasmids, making it an excellent tool for synthetic biologists.

The early diagnosis of active hepatitis C virus (HCV) infection remains a significant barrier to the treatment of the disease and to preventing the associated significant morbidity and mortality seen, worldwide. Current testing is delayed due to the high cost, long turnaround times and high expertise needed in centralised diagnostic laboratories. Here we demonstrate a user-friendly, low-cost pan-genotypic assay, based upon reverse transcriptase loop mediated isothermal amplification (RT-LAMP). We developed a prototype device for point-of-care use, comprising a LAMP amplification chamber and lateral flow nucleic acid detection strips, giving a visually-read, user-friendly result in, Current HCV nucleic acid-based diagnosis is largely performed in centralised laboratories. Here, the authors present a pan-genotypic RNA assay, based on reverse transcriptase loop mediated isothermal amplification and develop a low-cost prototype paper-based lateral flow device for point-of-care use, providing a visually read result within 40 min.

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3&prime, overhangs. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution.

Since the advent of genetic engineering, Escherichia coli, the most widely studied prokaryotic model organism, and other bacterial species have remained at the forefront of biological research. These ubiquitous microorganisms play an essential role in deciphering complex gene regulation mechanisms, large-scale recombinant protein production, and lately the two emerging areas of biotechnology—synthetic biology and metabolic engineering. Among a myriad of factors affecting prokaryotic gene expression, judicious choice of promoter remains one of the most challenging and impactful decisions in many biological experiments. This review provides a comprehensive overview of the current state of bacterial promoter engineering, with an emphasis on its applications in heterologous protein production, synthetic biology and metabolic engineering. In addition to highlighting relevant advances in these fields, the article facilitates the selection of an appropriate promoter by providing pertinent guidelines and explores the development of complementary databases, bioinformatics tools and promoter standardization procedures. The review ends by providing a quick overview of other emerging technologies and future prospects of this vital research area.

QuickStep-Cloning is a novel molecular cloning technique that builds upon the concepts of asymmetric PCR and megaprimer-based amplification of whole plasmid. It was designed specifically to address the major drawbacks of previously reported cloning methods. The fully optimized protocol allows for a seamless integration of a long DNA fragment into any position within a plasmid of choice, in a time-efficient and cost-effective manner, without the need of a tedious DNA gel purification, a restriction digestion, and an enzymatic ligation. QuickStep-Cloning can be completed in less than 6 h, significantly faster than most of the existing cloning methods, while retaining high efficiency.

QuickStep-Cloning is a novel molecular cloning technique that builds upon the concepts of asymmetric PCR and megaprimer-based amplification of whole plasmid. It was designed specifically to address the major drawbacks of previously reported cloning methods. The fully optimized protocol allows for a seamless integration of a long DNA fragment into any position within a plasmid of choice, in a time-efficient and cost-effective manner, without the need of a tedious DNA gel purification, a restriction digestion, and an enzymatic ligation. QuickStep-Cloning can be completed in less than 6 h, significantly faster than most of the existing cloning methods, while retaining high efficiency.

Background Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. Result Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3′-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. Conclusion QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists. Electronic supplementary material The online version of this article (doi:10.1186/s13036-015-0010-3) contains supplementary material, which is available to authorized users.