Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort

2014 ◽  
Vol 4 (3) ◽  
pp. 317-331 ◽  
Author(s):  
Sabrina Hoebenreich ◽  
Felipe E. Zilly ◽  
Carlos G. Acevedo-Rocha ◽  
Matías Zilly ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Solid Phase ◽  
Gene Synthesis

Solid-Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution?

ChemBioChem ◽  
2018 ◽  
Vol 19 (19) ◽  
pp. 2023-2032 ◽  
Author(s):  
Aitao Li ◽  
Zhoutong Sun ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Solid Phase ◽  
Gene Synthesis ◽  
Mutant Library ◽  
Library Construction ◽  
The Future

scholarly journals A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging

2001 ◽  
Vol 6 (4) ◽  
pp. 219-223 ◽  
Author(s):  
John M. Joern ◽  
Takeshi Sakamoto ◽  
Akira Arisawa ◽  
Frances H. Arnold
Keyword(s):  
Image Analysis ◽  
Directed Evolution ◽  
High Throughput ◽  
Digital Imaging ◽  
Solid Phase ◽  
High Throughput Screen ◽  
Microplate Reader ◽  
Dihydrodiol Dehydrogenase ◽  
Dioxygenase Activity

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-di- hydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30,uM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

Combinatorial Mutagenesis Algorithms, Digital Imaging Spectroscopy, and Solid-Phase Assays for Directed Evolution

2003 ◽  
Author(s):  
Simon Delagrave ◽  
Edward Bylina ◽  
William Coleman ◽  
Steven Robles ◽  
Mary Yang ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Digital Imaging ◽  
Solid Phase ◽  
Imaging Spectroscopy ◽  
Combinatorial Mutagenesis

Solid-phase purification of gene synthesis products using magnetic beads

2008 ◽  
Author(s):  
Mo Chao Huang ◽  
Mo-Huang Li ◽  
Jackie Y. Ying
Keyword(s):  
Magnetic Beads ◽  
Solid Phase ◽  
Gene Synthesis

Optimized Automated Solid Phase Synthesis of Oligonucleotides and Derivatives

1998 ◽  
Vol 53 (9) ◽  
pp. 1051-1068 ◽  
Author(s):  
Gabriel Alvarado Urbina ◽  
Gerald Grübler ◽  
Angelika Weiler ◽  
Hartmut Echner ◽  
Stanka Stoeva ◽  
...  
Keyword(s):  
High Performance ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Laser Desorption ◽  
Gene Synthesis ◽  
Chain Reaction ◽  
Laser Desorption Mass Spectrometry ◽  
Phase Synthesis ◽  
Polymerase Chain

An optimized automated synthesizer is presented for assembling oligonucleotides, thiooligonucleotides and 5′-modified oligonucleotides including: chemical phosphorylation, multihydroxyl derivatization with a non-nucleosidic phosphoramidite. The incorporation of biotin, fluorescein and rhodamine phosphoramidites is described. The purification and structure determination of oligo-nucleotides was confirmed using high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and laser desorption mass spectrometry (LDMS). Several applications and confirming data will be presented for gene synthesis and polymerase chain reaction (PCR) experiments.

scholarly journals Solid Phase Assembly of Fully Protected Trinucleotide Building Blocks for Codon-Based Gene Synthesis

2019 ◽  
Vol 9 (11) ◽  
pp. 2199 ◽  
Author(s):  
Ruth Suchsland ◽  
Bettina Appel ◽  
Matthäus Janczyk ◽  
Sabine Müller
Keyword(s):  
Amino Acids ◽  
Solid Phase ◽  
Building Blocks ◽  
Disulfide Bridge ◽  
Solid Support ◽  
Gene Synthesis ◽  
Directed Mutagenesis ◽  
Reducing Conditions

The use of pre-formed trinucleotides, representing codons of the 20 canonical amino acids, for oligonucleotide-directed mutagenesis offers the advantage of controlled randomization and generation of “smart libraries”. We here present a method for the preparation of fully protected trinucleotides on solid phase. The key issue of our strategy is the linkage of the starting nucleoside to the solid support via a traceless disulfide linker. Upon trinucleotide assembly, the disulfide bridge is cleaved under reducing conditions, and the fully protected trinucleotide is released with a terminal 3′-OH group.

scholarly journals Widely applicable background depletion step enables transaminase evolution through solid-phase screening

2019 ◽  
Vol 10 (23) ◽  
pp. 5952-5958 ◽  
Author(s):  
Matteo Planchestainer ◽  
Eimear Hegarty ◽  
Christian M. Heckmann ◽  
Louise J. Gourlay ◽  
Francesca Paradisi
Keyword(s):  
Directed Evolution ◽  
Solid Phase ◽  
Industrial Applications

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications.

scholarly journals High‐Throughput Solid‐Phase Assay for Substrate Profiling and Directed Evolution of Transketolase

ChemBioChem ◽  
2021 ◽  
Author(s):  
Nazim Ocal ◽  
Aurélie Lagarde ◽  
Mélanie L'enfant ◽  
Franck Charmantray ◽  
Laurence Hecquet
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Solid Phase ◽  
Solid Phase Assay
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