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Biology subjects

Stirling, F.

Publications and source records attributed to Stirling, F..

4 recordsLinked to original sources

Enabling community-based metrology for wood-degrading fungi

Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi can enable more direct means of manufacture. However, such practices are often ad hoc and not readily reproducible. We sought to develop standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products. Specifically, we show that a widely-available and globally-regularized consumer product (Pringles) can support the growth of wood-degrading fungi, and that growth on Pringles can be correlated with growth on a fully-traceable and compositionally characterized substrate (NIST Reference Material 8492 Eastern Cottonwood Biomass). So established, five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay. Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial.

synthetic biology↗

Synthetic cassettes for pH-mediated sensing, counting and containment

AbstractAs 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 106. We integrate acidTRP with cryodeath to produce a two-factor containment system with a combined survival ratio of less than 1 in 1011 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.

synthetic biology↗

Rational Design of Evolutionarily Stable Microbial Kill Switches

The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One particularly 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 novel 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. In addition, cryodeath was shown to control the growth environment of a bacterial population, with an escape rate of less than 1 in 105 after ten days in vivo.

synthetic biology↗

Rapid genome recoding by iterative recombineering of synthetic DNA

Genome recoding will provide a deeper understanding of genetics and transform biotechnology. We bypass the reliance of previous genome recoding methods on site-specific enzymes and demonstrate a rapid recombineering based strategy for writing genomes by Stepwise Integration of Rolling Circle Amplified Segments (SIRCAS). We installed the largest number of codon substitutions in a single organism yet published, creating a strain of Salmonella typhimurium with 1557 leucine codon changes across 200 kb of the genome.

synthetic biology↗