Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ ...engineered microorganisms in open-environment applications. Genetically encoded biocontainment systems may solve this issue. Here, we describe such a system based on conditional stability of essential proteins. We used a destabilizing domain degron stabilized by estradiol addition (ERdd). We ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of <5×10
. Removal of its C-terminal domain decreased the escape frequency further to <10
. Being based on conditional protein stability, the presented approach is mechanistically orthogonal to previously reported genetic biocontainment systems.
Perfluoroalkyl substances (PFAS) in water, sediment and fish were analyzed from a subtropical river, Jiulong River in the southeast of China, to character the sources, seasonal variations, ...bioconcentration and potential risk. PFAS in water, sediment, muscle and liver tissues of fish ranged from 2.5 to 410 ng L−1, 0.24–1.9 ng g−1 dw, 25–100 and 35–1100 ng g−1 ww, respectively. Generally, perfluorohexanoic acid (PFHxA) was the dominant compound in water, while, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) were the dominant compounds in sediment and fish tissues. High concentrations of PFAS in water were found near the machinery manufacturing and paper packaging plants in the north branch of Jiulong River. PFAS during the dry season were significantly (P < 0.01) higher than that during the normal season and wet season. The Kd of PFAS increased with the carbon chain length, and perfluoroalkyl sulfonic acids (PFSAs) exhibited higher Kd values than perfluoroalkyl carboxylic acids (PFCAs), indicating these long chain PFAS tended to be adsorbed by sediment. Long chain PFAS exhibited high bioconcentration factors (BCFs), while short and medium carbon chain PFAS had weak bioconcentration capacity. The hazard ratios (HR) suggested that frequent consumption of river fish may pose health risks to local population.
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•PFAS exhibited different composition profiles in water, sediment and fish.•Industrial activities contributed PFAS to the Jiulong River.•Carbon chain length and functional groups affected Kd values.•PFAS tended to accumulate in the liver of fish.
Design of a synthetic yeast genome Richardson, Sarah M.; Mitchell, Leslie A.; Stracquadanio, Giovanni ...
Science,
03/2017, Letnik:
355, Številka:
6329
Journal Article
Recenzirano
Odprti dostop
We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome ...deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by “endoreduplication intercross.” Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.
Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. ...Here we report a recombinase-based combinatorial method (termed "SCRaMbLE-in") to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of β-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology.
SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction. Here we describe a ...reporter of SCRaMbLEd cells using efficient selection, termed ReSCuES, based on a loxP-mediated switch of two auxotrophic markers. We show that all randomly isolated clones contained rearrangements within the synthetic chromosome, demonstrating high efficiency of selection. Using ReSCuES, we illustrate the ability of SCRaMbLE to generate strains with increased tolerance to several stress factors, such as ethanol, heat and acetic acid. Furthermore, by analyzing the tolerant strains, we are able to identify ACE2, a transcription factor required for septum destruction after cytokinesis, as a negative regulator of ethanol tolerance. Collectively, this work not only establishes a generic platform to rapidly identify strains of interest by SCRaMbLE, but also provides methods to dissect the underlying mechanisms of resistance.
We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within
(
), ...encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the
transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.
...the newly synthesized Sc3.0 chromosomes could then be combined into a single yeast to obtain strains with multiple chromosomes 8, or alternatively, these chromosomes could be merged into a single ...large chromosome 9, 10. ...regulatory elements such as promoters and terminators should be replaced by functionally validated but completely artificial sequences, or sequences from other yeast species as proposed for the base Sc3.0 strain. ...genes can be clustered according to their functionality or arranged based on their chromosomal locations.
Biofoundries provide an integrated infrastructure to enable the rapid design, construction, and testing of genetically reprogrammed organisms for biotechnology applications and research. Many ...biofoundries are being built and a Global Biofoundry Alliance has recently been established to coordinate activities worldwide.
Historical records of polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were analyzed in two dated sediment cores (DH05 and DH11) ...collected from the continental shelf of the East China Sea (ECS) to investigate the influence of anthropogenic activities on marine sediment over the past century. The concentrations and fluxes of 15 PAHs were in the range of 28.6–96.5 ng g−1 and 7.6–35.2 ng cm−2 yr−1 in DH05 (1920s–2009), 18.8–76.4 ng g−1 and 13.9–30.9 ng cm−2 yr−1 in DH11 (1860s–2009). The sedimentary records of PAHs in the two cores generally reflected the economic development and energy consumption change in China. Identification of sources suggested that PAHs in ECS were predominantly from petrogenic origin and various combustion sources. A change of source from low- and moderate-temperature combustion to high-temperature combustion process was observed. Although a production ban of technical HCH and DDT was imposed in China in 1983, their sedimentary fluxes display increasing trends or strong rebounds from 1980s to 1990s as recorded in the core profiles. High proportions of DDD + DDE and γ-HCH suggested those OCPs mainly derived from early residuals. Temporal trends of PCBs presented relative high levels from 1970s to 1980s and high proportions of PCB congeners with 3–6 chlorines atoms indicated industrial sources.
•Three kinds of typical organic pollutants in and out of the shelf mud of ECS were analyzed and compared.•Temporal variations of PAHs and OCs were used to reveal the history of socio-economic developments in the east of China.•The vertical variations of these pollutants implied the strong influence of anthropogenic activities in ESC.
The sedimentary records of PAHs, OCPs (HCHs and DDTs) and PCBs in two dated sediment cores of East China Sea were investigated.
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