Handmade microfluidic device for biochemical applications in emulsion

Murzabaev, Marsel; Kojima, Takaaki; Mizoguchi, Takuro; Kobayashi, Isao; DeKosky, Brandon J.; Georgiou, George; Nakano, Hideo

doi:10.1016/j.jbiosc.2015.08.001

Cited by 3 publications

(1 citation statement)

References 28 publications

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“…An oil mixture was composed of light mineral oil (Sigma-Aldrich Corporation, St. Louis, MO, USA), 4.5 % sorbitan monooleate (Nacalai Tesque), and 0.5 % Triton ® X-100 (Nacalai Tesque), as previously described 62, 63 . The CF-TXTL reaction solutions were mixed with the oil mixture and tapped twenty times in microtubes (Maruemu Corporations, Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

Reconstitution of ribosome self-replication outside a living cell

Kosaka

Miyawaki

Mori

et al. 2022

Preprint

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Ribosome biogenesis, a recursive process of pre-existing ribosomes self-replicating nascent ones, is pivotal in the self-replication of life. In Escherichia coli, three ribosomal RNAs (rRNAs) are transcribed, and 54 ribosomal proteins (r-proteins) are synthesized by pre-existing ribosomes as structural components. They are cotranscriptionally assembled in a cooperative hierarchy under the support of ~100 accessory factors. The reconstitution of ribosome biogenesis outside a living cell is an essential goal to understand the self-replication of life. However, this goal could not have been achieved so far due to its complexity. Here, we report the successful in vitro reconstitution of the entire ribosome biogenesis process. We hypothesized that mimicking in vivo ribosome biogenesis could result in in vitro ribosome biogenesis. Specifically, we found that coactivating the transcription of an rRNA operon, as well as the transcription and translation of 54 r-protein genes encoding r-proteins, and the coordinated ribosomal assembly in a cytoplasm-mimicking reaction solution, resulted in highly efficient in vitro reconstitution of ribosome biogenesis. Our achievement represents a critical step toward revealing fundamental principles underlying the self-replication of life and creating self-replicating artificial cells. We also succeeded in engineering rRNA and r-proteins by only adding mutant ribosomal genes in the reaction, enabling high-throughput and unconstrained creation of artificial ribosomes with altered or enhanced functionality.

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Section: Methodsmentioning

confidence: 99%

Reconstitution of ribosome self-replication outside a living cell

Kosaka

Miyawaki

Mori

et al. 2022

Preprint

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Ultra-high-throughput analysis of functional biomolecules using in vitro selection and bioinformatics

Kojima

2020

Bioscience, Biotechnology, and Biochemistry

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Functional analysis of biomolecules, including nucleic acids and proteins, is important for understanding biological mechanisms in living cells such as gene expression and metabolism. To analyze diverse biomolecular functions, large-scale screening systems for biomolecules have been developed for various applications such as to improve enzyme activity and identify target binding molecules. One of these systems, the Bead Display system, utilizes emulsion technology and is a powerful tool for rapidly screening functional nucleic acids or proteins in vitro. Furthermore, an analytical pipeline that consists of genomic systematic evolution of ligands by exponential enrichment (gSELEX)-Seq, gene expression analysis, and bioinformatics was shown to be a robust platform for comprehensively identifying genes regulated by a transcription factor. This review provides an overview of the biomolecular screening methods developed to date.

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Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

Lindenburg

Huovinen

Wiel

et al. 2020

Nucleic Acids Research

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Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method—termed SpliMLiB for Split-and-Mix Library on Beads—was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.

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Handmade microfluidic device for biochemical applications in emulsion

Cited by 3 publications

References 28 publications

Reconstitution of ribosome self-replication outside a living cell

Reconstitution of ribosome self-replication outside a living cell

Ultra-high-throughput analysis of functional biomolecules using in vitro selection and bioinformatics

Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

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