Summary The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One useful but challenging situation is a switch between life and death depending on environment. Here are presented “essentializer” and “cryodeath” circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 105 after ten days of growth in the mammalian gut.
Terminal restriction fragment length polymorphism (TRF or T-RFLP) analysis and 16S rDNA sequence analysis from clone libraries were used to examine cyanobacterial diversity in three types of predominant soil crusts in an arid grassland. Total DNA was extracted from cyanobacteria-, lichen-, or moss-dominated crusts that represent different successional stages in crust development, and which contribute different amounts of carbon and nitrogen into the ecosystem. Cyanobacterial 16S rRNA genes were amplified by PCR using cyanobacteria-specific 16S rDNA primers. Both TRF and clone sequence analyses indicated that the cyanobacterial crust type is dominated by strains of Microcoleus vaginatus, but also contains other cyanobacterial genera. In the moss crust, M. vaginatus-related sequences were also the most abundant types, together with sequences from moss chloroplasts. In contrast, sequences obtained from the lichen crust were surprisingly diverse, representing numerous genera, but including only two from M. vaginatus relatives. By obtaining clone sequence information, we were able to infer the composition of many peaks observed in TRF profiles, and all peaks predicted for clone sequences were observed in TRF analysis. This study provides the first TRF analysis of biological soil crusts and the first DNA-based comparison of cyanobacterial diversity between lichen-, cyano- and moss-dominated crusts. Results indicate that for this phylogenetic group, TRF analysis, in conjunction with limited sequence analysis, can provide accurate information about the composition and relative abundance of cyanobacterial types in soil crust communities.
Graphical Abstract Highlights d Containment system with an escape frequency below the detection limit of 10 À11 d Evolutionary stability achieved through toxin-antitoxin balancing d Pulse counter developed that responds to the falling edge of a signal d Counter will not advance unless 2 distinct signals are registered
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