ALBERT

Description: We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of 〉60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of 〉150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

Description: Satellite radar altimetry observations are used to derive short wavelength gravity anomaly fields over the Persian Gulf and the Caspian Sea, where in situ and ship-borne gravity measurements have limited spatial coverage. In this study the retracking algorithm ‘Extrema Retracking’ (ExtR) was employed to improve sea surface height (SSH) measurements that are highly biased in the study regions due to land contaminations in the footprints of the satellite altimetry observations. ExtR was applied to the waveforms sampled by the five satellite radar altimetry missions: TOPEX/POSEIDON, JASON-1, JASON-2, GFO and ERS-1. Along-track slopes have been estimated from the improved SSH measurements and used in an iterative process to estimate deflections of the vertical, and subsequently, the desired gravity anomalies. The main steps of the gravity anomaly computations involve estimating improved SSH using the ExtR technique, computing deflections of the vertical from interpolated SSHs on a regular grid using a biharmonic spline interpolation and finally estimating gridded gravity anomalies. A remove–compute–restore algorithm, based on the fast Fourier transform, has been applied to convert deflections of the vertical into gravity anomalies. Finally, spline interpolation has been used to estimate regular gravity anomaly grids over the two study regions. Results were evaluated by comparing the estimated altimetry-derived gravity anomalies (with and without implementing the ExtR algorithm) with ship-borne free air gravity anomaly observations, and free air gravity anomalies from the Earth Gravitational Model 2008 (EGM2008). The comparison indicates a range of 3–5 mGal in the residuals, which were computed by taking the differences between the retracked altimetry-derived gravity anomaly and the ship-borne data. The comparison of retracked data with ship-borne data indicates a range in the root-mean-square-error (RMSE) between approximately 1.8 and 4.4 mGal and a bias between 0.4062 and 2.1413 mGal over different areas. Also a maximum RMSE of 4.4069 mGal, with a mean value of 0.7615 mGal was obtained in the residuals. An average improvement of 5.2746 mGal in the RMSE of the altimetry-derived gravity anomalies corresponding to 89.9 per cent was obtained after applying the ExtR post-processing.

Description: Satellite radar altimetry observations are used to derive short wavelength gravity anomaly fields over the Persian Gulf and the Caspian Sea, where in situ and ship-borne gravity measurements have limited spatial coverage. In this study the retracking algorithm ‘Extrema Retracking’ (ExtR) was employed to improve sea surface height (SSH) measurements that are highly biased in the study regions due to land contaminations in the footprints of the satellite altimetry observations. ExtR was applied to the waveforms sampled by the five satellite radar altimetry missions: TOPEX/POSEIDON, JASON-1, JASON-2, GFO and ERS-1. Along-track slopes have been estimated from the improved SSH measurements and used in an iterative process to estimate deflections of the vertical, and subsequently, the desired gravity anomalies. The main steps of the gravity anomaly computations involve estimating improved SSH using the ExtR technique, computing deflections of the vertical from interpolated SSHs on a regular grid using a biharmonic spline interpolation and finally estimating gridded gravity anomalies. A remove–compute–restore algorithm, based on the fast Fourier transform, has been applied to convert deflections of the vertical into gravity anomalies. Finally, spline interpolation has been used to estimate regular gravity anomaly grids over the two study regions. Results were evaluated by comparing the estimated altimetry-derived gravity anomalies (with and without implementing the ExtR algorithm) with ship-borne free air gravity anomaly observations, and free air gravity anomalies from the Earth Gravitational Model 2008 (EGM2008). The comparison indicates a range of 3–5 mGal in the residuals, which were computed by taking the differences between the retracked altimetry-derived gravity anomaly and the ship-borne data. The comparison of retracked data with ship-borne data indicates a range in the root-mean-square-error (RMSE) between approximately 1.8 and 4.4 mGal and a bias between 0.4062 and 2.1413 mGal over different areas. Also a maximum RMSE of 4.4069 mGal, with a mean value of 0.7615 mGal was obtained in the residuals. An average improvement of 5.2746 mGal in the RMSE of the altimetry-derived gravity anomalies corresponding to 89.9 per cent was obtained after applying the ExtR post-processing.

Description: Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.

Description: Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli . This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.

Description: Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP / attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.

Description: We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii . Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus . Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.

Description: RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.

Description: Targeted resequencing technologies have allowed for efficient and cost-effective detection of genomic variants in specific regions of interest. Although capture sequencing has been primarily used for investigating single nucleotide variants and indels, it has the potential to elucidate a broader spectrum of genetic variation, including copy number variants (CNVs). Various methods exist for detecting CNV in whole-genome and exome sequencing datasets. However, no algorithms have been specifically designed for contiguous target sequencing, despite its increasing importance in clinical and research applications. We have developed cnvCapSeq, a novel method for accurate and sensitive CNV discovery and genotyping in long-range targeted resequencing. cnvCapSeq was benchmarked using a simulated contiguous capture sequencing dataset comprising 21 genomic loci of various lengths. cnvCapSeq was shown to outperform the best existing exome CNV method by a wide margin both in terms of sensitivity (92.0 versus 48.3%) and specificity (99.8 versus 70.5%). We also applied cnvCapSeq to a real capture sequencing cohort comprising a contiguous 358 kb region that contains the Complement Factor H gene cluster. In this dataset, cnvCapSeq identified 41 samples with CNV, including two with duplications, with a genotyping accuracy of 99%, as ascertained by quantitative real-time PCR.

Description: With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in Escherichia coli . We created Deconvoluter—ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N -ethyl- N -nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing (‘forward genomics’) demonstrates a strategy that could similarly enable rapid screens in many other microbes.