A Highly Efficient Molecular Cloning Platform that Utilises a Small Bacterial Toxin Gene

Mok, Wendy W. K.; Li, Yingfu

doi:10.1002/cbic.201300033

Cited by 10 publications

(7 citation statements)

References 23 publications

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“…Moreover, toxins on their own can also have various applications. Indeed, selective cloning vectors containing variants of IbsC, a type I E. coli toxic peptide, have been engineered [53]. In addition, chemical modifications of PepA1 toxin dramatically increased its antibacterial potential and its stability in human serum while considerably reducing its human cell toxicity, implying that toxins can be transformed into potent antibiotics [27].…”

Section: -Concluding Remarks and Perspectivesmentioning

confidence: 99%

Linking bacterial type I toxins with their actions

Brielle

Pinel-Marie

Felden

2016

Current Opinion in Microbiology

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Bacterial type I toxin-antitoxin systems consist of stable toxin-encoding mRNAs whose expression is counteracted by unstable RNA antitoxins. Accumulating evidence suggests that these players belong to broad regulatory networks influencing overall bacterial physiology. The majority of known transmembrane type I toxic peptides have conserved structural characteristics. However, recent studies demonstrated that their mechanisms of toxicity are diverse and complex. To better assess the current state of the art, type I toxins can be grouped into two classes according to their location and mechanisms of action: membrane-associated toxins acting by pore formation and/or by nucleoid condensation; and cytosolic toxins inducing nucleic acid cleavage. This classification will evolve as a result of future investigations.

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Section: -Concluding Remarks and Perspectivesmentioning

confidence: 99%

Linking bacterial type I toxins with their actions

Brielle

Pinel-Marie

Felden

2016

Current Opinion in Microbiology

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“…In this study, a 19-amino-acid toxin protein (IbsC) was applied as a reporter, which is highly hydrophobic and anchored in the inner membrane of E. coli . Overexpression of this protein can compromise the membrane's integrity and result in its depolarization (32–34). Extensive mutation can be tolerated by IbsC without loss of toxicity, and fusion of other proteins at its N terminus could be feasible without toxicity compromising.…”

Section: Resultsmentioning

confidence: 99%

Intracellular selection of trans-cleaving hammerhead ribozymes

Huang

Zhao

et al. 2019

Nucleic Acids Research

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Hammerhead ribozyme is the smallest and best characterized catalytic RNA-cleaving ribozyme. It has been reported as potential therapeutic tools to manipulate the expression of target genes. However, most of naturally occurring hammerhead ribozymes process self-cleavage rather than cleave substrate RNA in trans, and its high intracellular activity relies on the tertiary interaction of Loop II and steam I bulge, resulting in decreased performance as applied in gene silencing. We described a direct intracellular selection method to evolve hammerhead variants based on trans-cleavage mode via using a toxin gene as the reporter. And a dual fluorescence proteins system has also been established to quantitatively evaluate the efficiency of selected ribozymes in the cell. Based on this selection strategy, we obtained three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme. The best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead ribozyme. These observations imply the efficiency of the intracellular selection method of the trans-acting ribozyme and the potentials of improved ribozyme variants for research and therapeutic purposes.

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“…Although the precise mechanisms for their action are not well understood, these lethal genes should theoretically function properly in the IRDL cloning system. Recently, Mok and Li [36] developed a similar cloning method, referred to as the detox system, but they did not combine the digestion and ligation protocols into a single step. Therefore, the resulting procedure is 20 times as long as the IRDL cloning procedure (30 min digestion and 70 min ligation for detox cloning versus 5 min digestion and ligation for IRDL cloning).…”

Section: Discussionmentioning

confidence: 99%

IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

Wang

Liu

2014

PLoS ONE

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Rapid and efficient construction of expression vectors and subsequent transformation are basic recombinant methods for the investigation of gene functionality. Although novel cloning methods have recently been developed, many laboratories worldwide continue to use traditional restriction digestion-ligation methods to construct expression vectors owing to financial constraints and the unavailability of appropriate vectors. We describe an improved restriction digestion-ligation (IRDL) cloning method that combines the advantage of directional cloning from double digestion-ligation with that of a low background observed by using a positive selection marker gene ccdB to facilitate digestion and ligation in a single tube. The IRDL cloning overcomes the time-consuming and laborious limits of traditional methods, thereby providing an easy-to-use, low-cost, and one-step strategy for directional cloning of target DNA fragments into an expression vector. As a proof-of-concept example, we developed two yeast vectors to demonstrate the feasibility and the flexibility of the IRDL cloning method. This method would provide an effective and easy-to-use system for gene cloning and functional genomics studies.

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A Highly Efficient Molecular Cloning Platform that Utilises a Small Bacterial Toxin Gene

Cited by 10 publications

References 23 publications

Linking bacterial type I toxins with their actions

Linking bacterial type I toxins with their actions

Intracellular selection of trans-cleaving hammerhead ribozymes

IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

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