A modular autoinduction device for control of gene expression in Bacillus subtilis

Intense synthesis of proteins and chemicals in engineered microbes impose metabolic burden, frequently leading to reduced growth and heterogeneous cell population. Thus, the correct balance between growth and production is important. Such balance can be engineered through dynamic control of pathways, but few broadly applicable tools are available to achieve this. We present an autonomous control of gene expression mediated by quorum sensing in Bacillus subtilis, able to self-monitor and induce expression without human supervision. Two variations of the induction module and seven of the response module were engineered generating a range of induction folds and strengths for gene expression control. Our strongest response promoter is 2.5 and 3.2 times stronger than the well-characterized promoters P_srfA and P_veg, respectively. We applied our strongest autoinduction device for the production of the vitamin B₂. This study presents a toolbox of autoinduction modules for B. subtilis that is modular and tunable.     

Signatures of adaptation and genetic structure among the mainland populations of Pinus radiata (D. Don) inferred from SNP loci

Insights into the relative contributions of locus specific and genome-wide effects on population genetic diversity can be gained through separation of their resulting genetic signals. Here we explore patterns of adaptive and neutral genetic diversity in the disjunct natural populations of Pinus radiata (D. Don) from mainland California. A first-generation common garden of 447 individuals revealed significant differentiation of wood phenotypes among populations (P _ST), possibly reflecting local adaptation in response to environment. We subsequently screened all trees for genetic diversity at 149 candidate gene single nucleotide polymorphism (SNP) loci for signatures of adaptation. Ten loci were identified as being possible targets of diversifying selection following F _ST outlier tests. Multivariate canonical correlation performed on a data set of 444 individuals identified significant covariance between environment, adaptive phenotypes and outlier SNP diversity, lending support to the case for local adaptation suggested from F _ST and P _ST tests. Covariation among discrete sets of outlier SNPs and adaptive phenotypes (inferred from multivariate loadings) with environment are supported by existing studies of candidate gene function and genotype–phenotype association. Canonical analyses failed to detect significant correlations between environment and 139 non-outlier SNP loci, which were applied to estimate neutral patterns of genetic differentiation among populations (F _ST 4.3 %). Using this data set, significant hierarchical structure was detected, indicating three populations on the mainland. The hierarchical relationships based on neutral SNP markers (and SSR) were in contrast with those inferred from putatively adaptive loci, potentially highlighting the independent action of selection and demography in shaping genetic structure in this species.     

Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.     

Novel Method for Genomic Promoter Shuffling by Using Recyclable Cassettes

Genetic elements of interest can be introduced into the Saccharomyces cerevisiae genome via homologous recombination. The current method is to link such an element to a selectable marker gene to be integrated into the target locus. However, the marker gene in this method cannot be reused, which limits repeated manipulation of the yeast genome. An alternative method is to utilize a counterselectable gene, such as URA3, with flanking tandem repeats. After integration, URA3 along with one copy of the repeat can be popped out via internal recombination, leaving behind one copy of the unwanted repeat. Here we describe a novel concept of genetic element shuffling in which the tandem repeats are made of the desired genetic element, so that after integration and popping out, only one copy of the element remains at the desired locus to function. As a proof of principle, we constructed three recyclable cassettes (P_PGK1-URA3-P_PGK1, P_GAL1-URA3-P_GAL1, and P_tetO7-URA3-P_tetO7) and integrated them upstream of an engineered chromosomal P_HIS3-mCherry-Myc locus. After promoter shuffling, the mCherry-Myc gene was regulated precisely as anticipated.     

New Vectors for Chromosomal Integration Enable High-Level Constitutive or Inducible Magnetosome Expression of Fusion Proteins in Magnetospirillum gryphiswaldense

The alphaproteobacterium Magnetospirillum gryphiswaldense biomineralizes magnetosomes, which consist of monocrystalline magnetite cores enveloped by a phospholipid bilayer containing specific proteins. Magnetosomes represent magnetic nanoparticles with unprecedented magnetic and physicochemical characteristics. These make them potentially useful in a number of biotechnological and biomedical applications. Further functionalization can be achieved by expression of foreign proteins via genetic fusion to magnetosome anchor peptides. However, the available genetic tool set for strong and controlled protein expression in magnetotactic bacteria is very limited. Here, we describe versatile vectors for either inducible or high-level constitutive expression of proteins in M. gryphiswaldense. The combination of an engineered native P_mamDC promoter with a codon-optimized egfp gene (Mag-egfp) resulted in an 8-fold increase in constitutive expression and in brighter fluorescence. We further demonstrate that the widely used P_tet promoter is functional and tunable in M. gryphiswaldense. Stable and uniform expression of the EGFP and β-glucuronidase (GusA) reporters was achieved by single-copy chromosomal insertion via Tn5-mediated transposition. In addition, gene duplication by Mag-EGFP–EGFP fusions to MamC resulted in further increased magnetosome expression and fluorescence. Between 80 and 210 (for single MamC–Mag-EGFP) and 200 and 520 (for MamC–Mag-EGFP–EGFP) GFP copies were estimated to be expressed per individual magnetosome particle.