Search bioRxiv⌕ Search

Biology subjects

Ngo, P. H. T.

Publications and source records attributed to Ngo, P. H. T..

2 recordsLinked to original sources

Changes in coding and efficiency through modular modifications to a One Pot PURE system for in vitro transcription & translation

The incorporation of unnatural amino acids are attractive methods for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. In this work, we further adapted and modified the One Pot PURE for a robust and modular system of enzymatic single site-specific of unnatural amino acid. We demonstrated the flexibility of this system through the introduction of two orthogonal aminoacyl tRNA synthetases and the suppression of two distinctive stop codons.

synthetic biology↗

Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions

DNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 {degrees}C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

bioengineering↗