Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO2 capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO2, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO2, avoiding fossil carbon or competition with food production

A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer

Type IV pili-independent photocurrent production by the cyanobacterium Synechocystis sp. PCC 6803

Biophotovoltaic devices utilize photosynthetic organisms such as the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) to generate current for power or hydrogen production from light. These devices have been improved by both architecture engineering and genetic engineering of the phototrophic organism. However, genetic approaches are limited by lack of understanding of cellular mechanisms of electron transfer from internal metabolism to the cell exterior. Type IV pili have been implicated in extracellular electron transfer (EET) in some species of heterotrophic bacteria. Furthermore, conductive cell surface filaments have been reported for cyanobacteria, including Synechocystis. However, it remains unclear whether these filaments are type IV pili and whether they are involved in EET. Herein, a mediatorless electrochemical setup is used to compare the electrogenic output of wild-type Synechocystis to that of a ΔpilD mutant that cannot produce type IV pili. No differences in photocurrent, i.e., current in response to illumination, are detectable. Furthermore, measurements of individual pili using conductive atomic force microscopy indicate these structures are not conductive. These results suggest that pili are not required for EET by Synechocystis, supporting a role for shuttling of electrons via soluble redox mediators or direct interactions between the cell surface and extracellular substrates

Simmer analysis of prompt burst energetics experiments

The Prompt Burst Energetics experiments are designed to measure the pressure behavior of fuel and coolant as working fluids during a hypothetical prompt burst disassembly in an LMFBR. The work presented in this report consists of a parametric study of PBE-5S, a fresh oxide fuel experiment, using SIMMER-II. The various pressure sources in the experiment are examined, and the dominant source identified as incondensable contaminant gasses in the fuel. The important modeling uncertainties and limitations of SIMMER-II as applied to these experiments are discussed

Heat-transfer characteristics of dry porous particulate beds with internal heat generation. [PWR; BWR]

A combined experimental and analytical program has been undertaken to study the thermal characteristics of dry debris beds especially at high temperatures where radiative heat transfer contributes. Experiments have been conducted in-pile using intrinsic fission heating of UO/sub 2/ to simulate decay heat power levels. Both pure UO/sub 2/ and mixed UO/sub 2//steel beds in a helium atmosphere have been studied. Temperatures as high as 3100 K have been attained. Theremocouples and ultrasonic thermometers have been used to measure the bed temperatures. Also recent post-test metallographic examinations have revealed useful information on the materials interactions which can be correlated to changes in the thermal behavior of the bed. In addition to the experiments, a number of porous medium thermal conductivity models have been evaluated. From these, two models representing upper and lower bounds for the conductivity, in conjunction with a two-dimensional heat transfer analysis have been compared to the data

The Travels of John Magee:Tracing the Geographies of Britain's Itinerant Print-Sellers, 1789–1815

This article is concerned with the circulation of topical print culture in Britain during the Napoleonic Wars, focusing on carriers rather than texts. Centred on the biographical and autobiographical accounts of ballad singers and chapmen, especially John Magee's Travels, it maps the geographies of numerous print-selling itinerants. By stressing the role of these individuals as mediators of topical material, it argues that our reading of the reception of news, in an age of propaganda, should privilege the agency of transmitters as much as that of writers. The article's geographies also challenge a London-centric model for the diffusion of topical material