We describe a directed genome-engineering approach that combines genome-wide methods for mapping genes to traits [Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LBA, Gill RT (2010) Nat Biotechnol 28:856–862] with strategies for rapidly creating combinatorial ribosomal binding site (RBS) mutation libraries containing billions of targeted modifications [Wang HH, et al. (2009) Nature 460:894–898]. This approach should prove broadly applicable to various efforts focused on improving production of fuels, chemicals, and pharmaceuticals, among other products. We used barcoded promoter mutation libraries to map the effect of increased or decreased expression of nearly every gene in Escherichia coli onto growth in several model environments (cellulosic hydrolysate, low pH, and high acetate). Based on these data, we created and evaluated RBS mutant libraries (containing greater than 100,000,000 targeted mutations), targeting the genes identified to most affect growth. On laboratory timescales, we successfully identified a broad range of mutations (>25 growth-enhancing mutations confirmed), which improved growth rate 10–200% for several different conditions. Although successful, our efforts to identify superior combinations of growth-enhancing genes emphasized the importance of epistatic interactions among the targeted genes (synergistic, antagonistic) for taking full advantage of this approach to directed genome engineering.

Genome engineering of cyanobacteria is a promising area of development in order to produce fuels, feedstocks, and value-added chemicals in a sustainable way. Unfortunately, the current state of genome engineering tools for cyanobacteria lags far behind those of model organisms such as Escherichia coli and Saccharomyces cerevisiae. In this review, we present the current state of synthetic biology tools for genome engineering efforts in the most widely used cyanobacteria strains and areas that need concerted research efforts to improve tool development. Cyanobacteria pose unique challenges to genome engineering efforts because their cellular biology differs significantly from other eubacteria; therefore, tools developed for other genera are not directly transferrable. Standardized parts, such as promoters and ribosome binding sites, which control gene expression, require characterization in cyanobacteria in order to have fully predictable results. The application of these tools to genome engineering efforts is also discussed; the ability to do genome-wide searching and to introduce multiple mutations simultaneously is an area that needs additional research in order to enable fast and efficient strain engineering.

Abstract:In an attempt to recreate the microenvironment necessary for directed hematopoietic stem cell differentiation, control over the amount of ions available to the cells is necessary. The release of potassium ion and calcium ion via the control of cross-linking density of a poly(2-hydroxyethyl methacrylate) (pHEMA)-based hydrogel containing 1 mol % 2-methacryloyloxyethyl phosphorylcholine (MPC) and 5 mol % oligo(ethylene glycol) (400) monomethacrylate [OEG(400)MA] was investigated. Tetra(ethylene glycol) diacrylate (TEGDA), the cross-linker, was varied over the range of 1-12 mol %. Hydrogel discs (ϕ = 4.5 mm and h = 2.0 mm) were formed by UV polymerization within silicone isolators to contain 1.0 M CaCl 2 and 0.1 M KCl, respectively. Isothermal release profiles, were measured at 37 °C in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid sodium salt (HEPES) buffer using either calcium ion or potassium ion selective electrodes (ISE). The resulting release OPEN ACCESSPolymers 2013, 5 1242 profiles were found to be independent of cross-linking density. Average (n = 3) release profiles were fit to five different release models with the Korsmeyer-Peppas equation, a porous media transport model, exhibiting the greatest correlation (R 2 > 0.95). The diffusion exponent, n was calculated to be 0.24 ± 0.02 and 0.36 ± 0.04 for calcium ion and potassium ion respectively indicating non-Fickian diffusion. The resulting diffusion coefficients were calculated to be 2.6 × 10 −6 and 11.2 × 10 −6 cm 2 /s, which compare well to literature values of 2.25 × 10 −6 and 19.2 × 10 −6 cm 2 /s for calcium ion and potassium ion, respectively.

In response to demands for sustainable domestic fuel sources, research into biofuels has become increasingly important. Many challenges face biofuels in their effort to replace petroleum fuels, but rational strain engineering of algae and photosynthetic organisms offers a great deal of promise. For decades, mutations and stress responses in photosynthetic microbiota were seen to result in production of exciting high-energy fuel molecules, giving hope but minor capability for design. However, '-omics' techniques for visualizing entire cell processing has clarified biosynthesis and regulatory networks. Investigation into the promising production behaviors of the model organism C. reinhardtii and its mutants with these powerful techniques has improved predictability and understanding of the diverse, complex interactions within photosynthetic organisms. This new equipment has created an exciting new frontier for high-throughput, predictable engineering of photosynthetically produced carbon-neutral biofuels.