Biology subjects

Sharon, J.

Publications and source records attributed to Sharon, J..

3 recordsLinked to original sources

bioRxiv2025

One-pot cloning and protein expression platform for genetic engineering

In this work, we present a streamlined one-pot cloning and protein expression platform that integrates mutagenesis, plasmid assembly, and functional protein testing in a single reaction. By combining Golden Gate cloning with cell-free transcription-translation, we demonstrate efficient generation and screening of genetic variants without the need for intermediate purification or bacterial amplification. Using fluorescent proteins, luciferase enzymes, antibiotic-converting enzymes, and the violacein biosynthetic pathway, we validate the versatility of this approach for single-and multi-site mutagenesis, combinatorial variant libraries, metabolic pathway programming, and whole-plasmid assembly. By demonstrating compatibility with multiplexed reactions and multi-cistronic constructs, we establish this approach as a generalizable and automatable method for high-throughput cloning and protein engineering in synthetic biology.

bioengineering↗

bioRxiv2021

Akaby - cell-free protein expression system for linear templates

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.

synthetic biology↗

bioRxiv2021

T7Max transcription system

Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. Here we present a modified T7 RNA polymerase promoter that acts to significantly increase the yields of both transcription and translation within in vitro systems. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase-based in vitro protein expression system. Unlike other methods of limiting linear template degradation, the T7Max promoter increases transcript concentration in a T7 transcription reaction, providing more mRNA for translation.

synthetic biology↗