Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions.

The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits.M. J. and J. W. A. are supported by EPSRC.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/1751-7915.1229

High molecular weight DNA assembly in vivo for synthetic biology applications.

DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.Cordial thanks to all researchers who contributed to these advances in DNA assembly. M.J. and J.W.A. are supported by the Engineering and Physical Sciences Research Council (EPSRC).This is the final version of the article. It first appeared from Taylor & Francis via https://doi.org/10.3109/07388551.2016.114139

Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.M.J. and J.W.A are supported by EPSRC, OpenPlant Fund and SynBio Fund.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.mimet.2016.03.01

Empirical Bayes method for reducing false discovery rates of correlation matrices with block diagonal structure

$\textbf{Background:}$ Correlation matrices are important in inferring relationships and networks between regulatory or signalling elements in biological systems. With currently available technology sample sizes for experiments are typically small, meaning that these correlations can be difficult to estimate. At a genome-wide scale estimation of correlation matrices can also be computationally demanding. $\textbf{Results:}$ We develop an empirical Bayes approach to improve covariance estimates for gene expression, where we assume the covariance matrix takes a block diagonal form. Our method shows lower false discovery rates than existing methods on simulated data. Applied to a real data set from $\textit{Bacillus subtilis}$ we demonstrate it's ability to detecting known regulatory units and interactions between them. $\textbf{Conclusions:}$ We demonstrate that, compared to existing methods, our method is able to find significant covariances and also to control false discovery rates, even when the sample size is small ($n$=10). The method can be used to find potential regulatory networks, and it may also be used as a pre-processing step for methods that calculate, for example, partial correlations, so enabling the inference of the causal and hierarchical structure of the networks.CP is funded by the Engineering and Physical Sciences Research Council (EPSRC) doctoral program

Quality determination and the repair of poor quality spots in array experiments.

BACKGROUND: A common feature of microarray experiments is the occurrence of missing gene expression data. These missing values occur for a variety of reasons, in particular, because of the filtering of poor quality spots and the removal of undefined values when a logarithmic transformation is applied to negative background-corrected intensities. The efficiency and power of an analysis performed can be substantially reduced by having an incomplete matrix of gene intensities. Additionally, most statistical methods require a complete intensity matrix. Furthermore, biases may be introduced into analyses through missing information on some genes. Thus methods for appropriately replacing (imputing) missing data and/or weighting poor quality spots are required. RESULTS: We present a likelihood-based method for imputing missing data or weighting poor quality spots that requires a number of biological or technical replicates. This likelihood-based approach assumes that the data for a given spot arising from each channel of a two-dye (two-channel) cDNA microarray comparison experiment independently come from a three-component mixture distribution--the parameters of which are estimated through use of a constrained E-M algorithm. Posterior probabilities of belonging to each component of the mixture distributions are calculated and used to decide whether imputation is required. These posterior probabilities may also be used to construct quality weights that can down-weight poor quality spots in any analysis performed afterwards. The approach is illustrated using data obtained from an experiment to observe gene expression changes with 24 hr paclitaxel (Taxol) treatment on a human cervical cancer derived cell line (HeLa). CONCLUSION: As the quality of microarray experiments affect downstream processes, it is important to have a reliable and automatic method of identifying poor quality spots and arrays. We propose a method of identifying poor quality spots, and suggest a method of repairing the arrays by either imputation or assigning quality weights to the spots. This repaired data set would be less biased and can be analysed using any of the appropriate statistical methods found in the microarray literature.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Characterization of Intrinsic Properties of Promoters.

Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic contexts of the measurement. Furthermore, in vivo measurement methods must accommodate variation in translation, protein folding, and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered to be extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents, and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method to quantitatively describe promoter properties.TJR was supported by a Microsoft Research studentship and EC FP7 Project No. 612146 (PLASWIRES) awarded to JH, JRB by a Microsoft Research studentship and internship, and FF by CONICYT-PAI/Concurso Nacional de Apoyo al Retorno de Investigadores/as desde el Extranjero Folio 8213002 7, and EPSRC grant EP/H019162/1 awarded to JH. JWA acknowledges the EPSRC and the Wellcome Trust for support.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acssynbio.5b0011

Epigenomic Modifications Predict Active Promoters and Gene Structure in Toxoplasma gondii

Mechanisms of gene regulation are poorly understood in Apicomplexa, a phylum that encompasses deadly human pathogens like Plasmodium and Toxoplasma. Initial studies suggest that epigenetic phenomena, including histone modifications and chromatin remodeling, have a profound effect upon gene expression and expression of virulence traits. Using the model organism Toxoplasma gondii, we characterized the epigenetic organization and transcription patterns of a contiguous 1% of the T. gondii genome using custom oligonucleotide microarrays. We show that methylation and acetylation of histones H3 and H4 are landmarks of active promoters in T. gondii that allow us to deduce the position and directionality of gene promoters with >95% accuracy. These histone methylation and acetylation “activation” marks are strongly associated with gene expression. We also demonstrate that the pattern of histone H3 arginine methylation distinguishes certain promoters, illustrating the complexity of the histone modification machinery in Toxoplasma. By integrating epigenetic data, gene prediction analysis, and gene expression data from the tachyzoite stage, we illustrate feasibility of creating an epigenomic map of T. gondii tachyzoite gene expression. Further, we illustrate the utility of the epigenomic map to empirically and biologically annotate the genome and show that this approach enables identification of previously unknown genes. Thus, our epigenomics approach provides novel insights into regulation of gene expression in the Apicomplexa. In addition, with its compact genome, genetic tractability, and discrete life cycle stages, T. gondii provides an important new model to study the evolutionarily conserved components of the histone code

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation

The Escherichia coli motA flagellar gene as a potential integration site for large synthetic DNA

Escherichia coli is used as a chassis for many synthetic biology applications. However, the limitations of maintaining recombinant plasmids extra-chromosomally include increased metabolic burden to the host, constant selective pressure, variable plasmid copy number and plasmid instability that leads to curing. Hence, to overcome these limitations, DNA constructs are integrated into the bacterial chromosome to allow stable control of copy number and to reduce the metabolic burden towards the surrogate host. Non-essential E. coli flagellar genes have been proposed as potential chromosomal insertion target sites. In this study, we validated and compared the efficiency of two loci, namely motA and flgG, as target sites for synthetic biology applications. To enable this comparison, a dual reporter strain (DRS) that utilises two reporter proteins, EforRED and Venus, was developed as a test case. Initially, a yellow reporter plasmid k14.1_Venus was constructed and subsequently used as the plasmid backbone for the generation of two other plasmids, k14.1_eforRED and pcat_Venus, required to build the dual reporter strain. In the DRS, the eforRED gene was inserted into flgG whereas motA was disrupted by Venus. This mutant strain was defective in motility (p<0.001) but growth rate was unaffected. The fluorescence emitted by Venus was higher (p<0.05) compared to EforRED, suggesting that motA is the better chromosomal target locus compared to flgG. Hence, this study proposes the use of E. coli motA as the site for chromosomal insertion for future synthetic biology applications

Antiplasmodial and trypanocidal activity of violacein and deoxyviolacein produced from synthetic operons.

BACKGROUND: Violacein is a deep violet compound that is produced by a number of bacterial species. It is synthesized from tryptophan by a pathway that involves the sequential action of 5 different enzymes (encoded by genes vioA to vioE). Violacein has antibacterial, antiparasitic, and antiviral activities, and also has the potential of inducing apoptosis in certain cancer cells. RESULTS: Here, we describe the construction of a series of plasmids harboring the complete or partial violacein biosynthesis operon and their use to enable production of violacein and deoxyviolacein in E.coli. We performed in vitro assays to determine the biological activity of these compounds against Plasmodium, Trypanosoma, and mammalian cells. We found that, while deoxyviolacein has a lower activity against parasites than violacein, its toxicity to mammalian cells is insignificant compared to that of violacein. CONCLUSIONS: We constructed E. coli strains capable of producing biologically active violacein and related compounds, and propose that deoxyviolacein might be a useful starting compound for the development of antiparasite drugs