Simple method to extract extinction coefficients of films with the resolution of 10-5 using just transmission data and application to intermolecular charge-transfer absorption in an exciplex-forming organic film.

A simple method is presented to determine the thickness, refractive index and extinction coefficient of a film with the resolutions of ±1 nm, ±2 × 10-3, and 6 × 10-5, respectively. The method requires only ultraviolet-visible-near-infrared spectroscopy measurement of the transmittance of films with thicknesses of a few micrometers. A very small extinction coefficient of intermolecular charge transfer (CT) absorption of an exciplex-forming organic film is measured in the sub-bandgap wavelength region. This is to demonstrate that the CT absorption with extinction coefficient in the range of 10-3 to 10-5 can be measured indeed using the method, which is in the same resolution as photothermal deflection spectroscopy and Fourier transform photocurrent spectroscopy. The simplicity and feasibility of the proposed approach is expected to promote active study of intermolecular CT absorption in exciplex-forming films.

Highly selective retrieval of accurate DNA utilizing a pool of in situ-replicated DNA from multiple next-generation sequencing platforms.

Scalable and cost-effective production of error-free DNA is critical to meet the increased demand for such DNA in the field of biological science. Methods based on 'Dial-out PCR' have enabled the high-throughput error-free DNA synthesis from a microarray-synthesized DNA pool by labeling with retrieval PCR tags, and retrieving error-free DNA of which the sequence is identified via next generation sequencing (NGS). However, most of the retrieved products contain byproducts due to background amplification of redundantly labeled DNAs. Here, we present a highly selective retrieval method of desired DNA from a pool of millions of DNA clones from NGS platforms. Our strategy is based on replicating entire sequence-verified DNA molecules from NGS plates to obtain population-controlled DNA pool. Using the NGS-replica pool, we could perform improved and selective retrieval of desired DNA from the replicated DNA pool compared to other dial-out PCR based methods. To evaluate the method, we tested this strategy by using 454, Illumina, and Ion Torrent platforms for producing NGS-replica pool. As a result, we observed a highly selective retrieval yield of over 95%. We anticipate that applications based on this method will enable the preparation of high-fidelity sequenced DNA from heterogeneous collections of DNA molecules.

Systematic Dissection of Sequence Elements Controlling σ70 Promoters Using a Genomically Encoded Multiplexed Reporter Assay in Escherichia coli.

Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In Escherichia coli, decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10898 σ70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our data set using a simple log-linear statistical model. Simple neural network models explain >95% of the variance in our data set by capturing nonlinear interactions with the spacer, background, and UP elements.

Genome-scale promoter engineering by coselection MAGE.

Multiplex automated genome engineering (MAGE) uses short oligonucleotides to scarlessly modify genomes; however, insertions >10 bases are still inefficient but can be improved substantially by selection of highly modified chromosomes. Here we describe 'coselection' MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Promoter libraries can be quickly generated to study gain-of-function epistatic interactions in gene networks.

'Shotgun DNA synthesis' for the high-throughput construction of large DNA molecules.

We developed a highly scalable 'shotgun' DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.

A multiplexed bacterial two-hybrid for rapid characterization of protein-protein interactions and iterative protein design.

Protein-protein interactions (PPIs) are crucial for biological functions and have applications ranging from drug design to synthetic cell circuits. Coiled-coils have been used as a model to study the sequence determinants of specificity. However, building well-behaved sets of orthogonal pairs of coiled-coils remains challenging due to inaccurate predictions of orthogonality and difficulties in testing at scale. To address this, we develop the next-generation bacterial two-hybrid (NGB2H) method, which allows for the rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way using next-generation sequencing readout. We design, build, and test large sets of orthogonal synthetic coiled-coils, assayed over 8,000 PPIs, and used the dataset to train a more accurate coiled-coil scoring algorithm (iCipa). After characterizing nearly 18,000 new PPIs, we identify to the best of our knowledge the largest set of orthogonal coiled-coils to date, with fifteen on-target interactions. Our approach provides a powerful tool for the design of orthogonal PPIs.

Multiple target loci assembly sequencing (mTAS).

Here we present multiple target loci assembly sequencing (mTAS), a method for examining multiple genomic loci in a single DNA sequencing read. The key to the success of mTAS target sequencing is the uniform amplification of multiple target genomic loci into a single DNA fragment using polymerase cycling assembly (PCA). Using this strategy, we successfully collected multiloci sequence information from a single DNA sequencing run. We applied mTAS to examine 29 different sets of human genomic loci, each containing from 2 to 11 single-nucleotide polymorphisms (SNP) present at different exons. We believe mTAS can be used to reduce the cost of Sanger sequencing-based genetic analysis.

A fluorescence selection method for accurate large-gene synthesis.

The fundamental problem for low-cost gene synthesis is errors that occur during the synthetic process. To address this problem, we developed a practical method that exploits the fact that the predominant errors are deletions. In this method, a simple fluorescence-based readout was used to distinguish error-free synthetic DNA molecules. To do this, we constructed vectors that contained multiple cloning sites and GFP. In the vectors, the GFP gene is designed to be out-of-frame, but insertion of an in-framed synthetic DNA construct into the appropriate cloning site will lead to fluorescent cell colonies. We successfully used this method to synthesize five genes and improved the bp per error from 629 to 6552 by selecting green fluorescent colonies.

A high-throughput optomechanical retrieval method for sequence-verified clonal DNA from the NGS platform.

Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering. DNA clones on next-generation sequencing (NGS) platforms have the potential to be a rich and cost-effective source of sequence-verified DNAs as a precursor for DNA writing. However, it is still very challenging to retrieve target clonal DNA from high-density NGS platforms. Here we propose an enabling technology called 'Sniper Cloning' that enables the precise mapping of target clone features on NGS platforms and non-contact rapid retrieval of targets for the full utilization of DNA clones. By merging the three cutting-edge technologies of NGS, DNA microarray and our pulse laser retrieval system, Sniper Cloning is a week-long process that produces 5,188 error-free synthetic DNAs in a single run of NGS with a single microarray DNA pool. We believe that this technology has potential as a universal tool for DNA writing in biological sciences.

A library of TAL effector nucleases spanning the human genome.

Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

Hierarchical gene synthesis using DNA microchip oligonucleotides.

High-cost of oligonucleotides is one of the major problems to low-cost gene synthesis. Although DNA oligonucleotides from cleavable DNA microchips has been adopted for the low-cost gene synthesis, construction of DNA molecules larger than 1 kb has been largely hampered due to the difficulties of DNA assembly associated with the negligible quantity of chip oligonucleotides. Here we report a hierarchical method for the synthesis of large genes using oligonucleotides from programmable DNA microchips. Using this hierarchical method, we successfully synthesized 1056 bp Dpo4 and 2325 bp Pfu DNA polymerase genes as models. This hierarchical strategy can be further expanded for the syntheses of multiple large genes in a scalable manner.