Towards developing algal synthetic biology.

The genetic, physiological and metabolic diversity of microalgae has driven fundamental research into photosynthesis, flagella structure and function, and eukaryotic evolution. Within the last 10 years these organisms have also been investigated as potential biotechnology platforms, for example to produce high value compounds such as long chain polyunsaturated fatty acids, pigments and antioxidants, and for biodiesel precursors, in particular triacylglycerols (TAGs). Transformation protocols, molecular tools and genome sequences are available for a number of model species including the green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, although for both species there are bottlenecks to be overcome to allow rapid and predictable genetic manipulation. One approach to do this would be to apply the principles of synthetic biology to microalgae, namely the cycle of Design-Build-Test, which requires more robust, predictable and high throughput methods. In this mini-review we highlight recent progress in the areas of improving transgene expression, genome editing, identification and design of standard genetic elements (parts), and the use of microfluidics to increase throughput. We suggest that combining these approaches will provide the means to establish algal synthetic biology, and that application of standard parts and workflows will avoid parallel development and capitalize on lessons learned from other systems.We thank the following for funding: the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK grant BB/I00680X/1 and the European Commission 7th Framework Program (FP7) project SPLASH (Sustainable 8 PoLymers from Algae Sugars and Hydrocarbons), grant agreement number 311956.This is the author accepted manuscript. The final version is available from Portland Press via http://dx.doi.org/10.1042/BST20160061

Analysis of prehistoric brown earth paleosols under the podzol soils of Exmoor, UK

The deforestation of the upland landscapes in southwest Britain during prehistory is an established archaeological narrative, documenting human impacts on the environment and questioning the relationship of prehistoric societies to the upland landscapes they inhabited. Allied to the paleoenvironmental analyses of pollen sequences, which have provided the evidence of this change, there has been some investigation of prehistoric paleosols fossilized under principally Bronze Age archaeological monuments. These analyses identified brown earth soils that were originally associated with temperate deciduous woodland, on occasion showing evidence of human impacts such as tilling. However, the number of analyses of these paleosols has been limited. This study presents the first analysis of a series of pre‐podzol brown earth paleosols on Exmoor, UK, two of which are associated with colluvial soil erosion sediments before the formation of peat. This study indicates these paleosols are spatially extensive and have considerable potential to inform a more nuanced understanding of prehistoric human impacts on the upland environments of the early‐mid Holocene and assess human agency in driving ecosystem change

Culture of airway epithelial cells from neonates sampled within 48-hours of birth

Peer reviewedPublisher PD

Role of riboswitches in gene regulation and their potential for algal biotechnology.

Riboswitches are regulatory elements in messenger RNA to which specific ligands can bind directly in the absence of proteins. Ligand binding alters the mRNA secondary structure, thereby affecting expression of the encoded protein. Riboswitches are widespread in prokaryotes, with over 20 different effector ligands known, including amino acids, cofactors, and Mg(2+) ions, and gene expression is generally regulated by affecting translation or termination of transcription. In plants, fungi, and microalgae, riboswitches have been found, but only those that bind thiamine pyrophosphate. These eukaryotic riboswitches operate by causing alternative splicing of the transcript. Here, we review the current status of riboswitch research with specific emphasis on microalgae. We discuss new riboswitch discoveries and insights into the underlying mechanism of action, and how next generation sequencing technology provides the motivation and opportunity to improve our understanding of these rare but important regulatory elements. We also highlight the potential of microalgal riboswitches as a tool for synthetic biology and industrial biotechnology.G.T.D.T.N was funded in part by Murray Edwards College and the Cambridge Philosophical Society. M.A.S was funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/I00680X/1 and the European Commission 7th Framework Programme (FP7) project SPLASH (Sustainable PoLymers from Algae Sugars and Hydrocarbons), grant agreement number 311956. K.E.H was funded by BBSRC grant BB/I013164/1.This is the author accepted manuscript. The final version is available from Wiley via https://doi.org/10.1111/jpy.1241

A national-scale seasonal hydrological forecast system: development and evaluation over Britain

Skilful winter seasonal predictions for the North Atlantic circulation and northern Europe have now been demonstrated and the potential for seasonal hydrological forecasting in the UK is now being explored. One of the techniques being used combines seasonal rainfall forecasts provided by operational weather forecast systems with hydrological modelling tools to provide estimates of seasonal mean river flows up to a few months ahead. The work presented here shows how spatial information contained in a distributed hydrological model typically requiring high-resolution (daily or better) rainfall data can be used to provide an initial condition for a much simpler forecast model tailored to use low-resolution monthly rainfall forecasts. Rainfall forecasts (“hindcasts”) from the GloSea5 model (1996 to 2009) are used to provide the first assessment of skill in these national-scale flow forecasts. The skill in the combined modelling system is assessed for different seasons and regions of Britain, and compared to what might be achieved using other approaches such as use of an ensemble of historical rainfall in a hydrological model, or a simple flow persistence forecast. The analysis indicates that only limited forecast skill is achievable for Spring and Summer seasonal hydrological forecasts; however, Autumn and Winter flows can be reasonably well forecast using (ensemble mean) rainfall forecasts based on either GloSea5 forecasts or historical rainfall (the preferred type of forecast depends on the region). Flow forecasts using ensemble mean GloSea5 rainfall perform most consistently well across Britain, and provide the most skilful forecasts overall at the 3-month lead time. Much of the skill (64 %) in the 1-month ahead seasonal flow forecasts can be attributed to the hydrological initial condition (particularly in regions with a significant groundwater contribution to flows), whereas for the 3-month ahead lead time, GloSea5 forecasts account for  ∼ 70 % of the forecast skill (mostly in areas of high rainfall to the north and west) and only 30 % of the skill arises from hydrological memory (typically groundwater-dominated areas). Given the high spatial heterogeneity in typical patterns of UK rainfall and evaporation, future development of skilful spatially distributed seasonal forecasts could lead to substantial improvements in seasonal flow forecast capability, potentially benefitting practitioners interested in predicting hydrological extremes, not only in the UK but also across Europe

Establishing Chlamydomonas reinhardtii as an industrial biotechnology host.

Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach.M.A.S and J.R were funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/I00680X/1, M.A.S was also funded by the European Commission 7th Framework Programme (FP7) project SPLASH (Sustainable PoLymers from Algae Sugars and Hydrocarbons), grant agreement number 311956. G.T.D.T.N was funded in part by Murray Edwards College and the Cambridge Philosophical Society. D.L. was funded by the Bill and Melinda Gates Foundation, and K.E.H was funded by BBSRC grant BB/I013164/1.This paper was originally published in The Plant Journal (Scaife MA, Nguyen GTDT, Rico J, Lambert D, Helliwell KE, Smith AG, The Plant Journal 2015, doi:10.1111/tpj.12781)

Primary Paediatric Bronchial Airway Epithelial Cell in Vitro Responses to Environmental Exposures

Acknowledgments: The authors are grateful to the children who participated in this study and their parents. The authors would like to thank the following colleagues in the department of Ear Nose and Throat Surgery at NHS Grampian for allowing us to recruit their patients: Nicola Kryle, Derek Veitch, Kim Ah-See, Bhaskar Ram, Sangeeta Maini and Clive Brewis. We are also grateful to Tenovus Scotland whose funds enabled analysis of the samples (Grant reference G13_17) but did not include open access publishing fees.Peer reviewedPublisher PD

Exploring the Impact of Terminators on Transgene Expression in <i>Chlamydomonas reinhardtii</i> with a Synthetic Biology Approach.

Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design

Green genes: bioinformatics and systems-biology innovations drive algal biotechnology.

Many species of microalgae produce hydrocarbons, polysaccharides, and other valuable products in significant amounts. However, large-scale production of algal products is not yet competitive against non-renewable alternatives from fossil fuel. Metabolic engineering approaches will help to improve productivity, but the exact metabolic pathways and the identities of the majority of the genes involved remain unknown. Recent advances in bioinformatics and systems-biology modeling coupled with increasing numbers of algal genome-sequencing projects are providing the means to address this. A multidisciplinary integration of methods will provide synergy for a systems-level understanding of microalgae, and thereby accelerate the improvement of industrially valuable strains. In this review we highlight recent advances and challenges to microalgal research and discuss future potential.We acknowledge support from the EU FP7 project SPLASH (Sustainable PoLymers from Algae Sugars and Hydrocarbons), grant agreement number 311956.This is the accepted manuscript. The final version is available from Cell/Elsevier at http://www.sciencedirect.com/science/article/pii/S016777991400196

Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas.

Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. Here we took a mutational approach to determine functionally important nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana. These chimeric riboswitches displayed a distinct specificity and dynamic range of responses to different ligands. Our studies demonstrate ease of assembly as 5'UTR DNA parts, predictability of output, and utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae.BBSR