How to model DNA replication in stochastic models of synthetic genetic circuits (and why)

Biocircuit modeling sometimes requires explicit tracking of a self-replicating DNA species. The most obvious, straightforward way to model a replicating DNA is structurally unstable and leads to pathological model behavior. We describe a simple, stable replication mechanism with good model behavior and show how to derive it from a mechanistic model of ColE1 replication.

Development of a novel T-vector supporting efficient green-white screening

T-vectors play an important role in cloning of polymerase chain reaction products. In the present study, a novel pUEG-T vector was developed using enhanced green fluorescent protein (EGFP) as an indicator. To improve EGFP-based green-white screening, the lipoprotein mutant promoter, a strong constitutive promoter, was utilized to control the expression of egfp gene. Two other efficient expression elements, the ColE1 replication origin of pUC18 and the expression cassette of pET-28a, were also integrated into pUEG-T vector. Expression analysis demonstrated the efficient accumulation of active EGFPs in Escherichia coli DH5α cells carrying the T-vector precursor pUEG. In T-A cloning using pUEG-T vector, white colonies containing foreign DNA and green colonies having no insertion could be handily distinguished under normal white light, without any chemical inducer or chromogenic substrate. Furthermore, no false positive was observed in any of the tested white colonies. This proves that pUEG-T is an inexpensive, convenient and efficient T-vector.

RNase/Anti-RNase Activities of the Bacterial parD Toxin-Antitoxin System

ABSTRACT The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single- and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.