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

As pH is fundamental to all biological processes, pH-responsive bacterial genetic circuits enable precise sensing in any environment. Where unintentional release of engineered bacteria poses a concern, coupling pH sensing to expression of a toxin creates an effective bacterial containment system. Here, we present a pH-sensitive kill switch (acidic Termination of Replicating Population; acidTRP), based on the E. coli asr promoter, with a survival ratio of less than 1 in 10 6 . We integrate acidTRP with cryodeath to produce a two-factor containment system with a combined survival ratio of less than 1 in 10 11 whilst maintaining evolutionary stability. We further develop a pulse-counting circuit with single cell readout for each administered stimulus pulse. We use this pulse-counter to record multiple pH changes and combine it with acidTRP to make a two-count acid-sensitive kill switch. These results demonstrate the ability to build complex genetic systems for biological containment.

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The grasping organ of Dendrocoelum lacteum is situated on the ventral surface of the anterior part of the head of this worm, between its two auricular appendages.Ijima ('84) records this organ as used for locomotion against currents. Gamble ('96, describes this species as " affixing a sucker, placed on the under side of the head, to the substratum, and pulling the posterior end close to this. The sucker, discovered by Leydig, is even better developed in (Plamria) punctata, P. mrazekii, and P. cavatica, and is an efficient adhering-organ, which has probably been developed from a similar but simpler structure found in a considerable number of both fresh-water and marine Triclads."How far these interpretations apply to Dendrocoelum lacteum, will appear in the following account. This paper is based on an experimental study of the function of the grasping organ in Dendrocoelum lacteum, and I wish to thank Dr G. H. Parker, under whom the work has been done, for his kind assistance.

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