methods-for-embedding-cell-free-protein-synthesis-reactions-macro

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...

Newcastle University

The on-column selective conversion of guanosine to thioguanosine (tG) yields modified oligomers that exhibit destabilisation over the fully complementary duplex. Restoration to a stabilised duplex is induced through thio-directed Cd coordination; a route for healing DNA damage. Short oligomers are G-specifically thiolated through a modified on-column protocol without the need for costly thioguanosine phosphoramidites. Addition of Cd ions to a duplex containing a highly disrupted tG central mismatch sequence, 3'-A tG T -5', suggests a (tG) Cd central coordination regime, resulting in increased base stacking and duplex stability. Equilibrium molecular dynamic calculations support the hypothesis of metal-induced healing of the thiolated duplex. The 2 nm displacement of the central tG mismatched region is dramatically reduced after the addition of a chemical stimuli, Cd ions, returning to a minimized fluctuational state comparable to the unmodified fully complementary oligomer.

Duplex Healing of Selectively Thiolated Guanosine Mismatches through a Cd Chemical Stimulus.

The synthesis of designer DNA requires an approach where the user can determine both the sequence and the number of nucleobases. The protocol outlined here describes an enzymatic method for the synthesis of long repeat-sequence DNA. The method utilizes a PCR-based approach; starting with short oligo-seeds, c.a. 20 bp, bearing a minimum of two repeating units of >8 bp sequences. During each heat-cool cycle, the oligo-seeds reanneal imperfectly leaving an overhang, which is then extended by the polymerase. The final length of the DNA is determined by the number of heat-cool cycles performed, reaching up to 20,000 bp after 20 cycles.

The Synthesis of Designer DNA.

The self-priming synthesis of multiply modified DNA by the extension of repeating unit duplex "oligoseeds" provides a source of versatile DNA. Sterically-demanding nucleotides 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP as well as 5-(octadiynyl)-dCTP were incorporated into two extending oligoseeds; [GATC] /[GATC] and [A G] /[CT ] . The products contained modifications on one or both strands of DNA, demonstrating their recognition by the polymerase as both template (reading) and substrate (writing). Nucleobase modifications that lie in the major groove were reliably read and written by the polymerase during the extension reaction, even when bulky or in contiguous sequences. Repeat sequence DNA over 500 bp long, bearing four different modified units was produced by this method. The number, position and type of modification, as well as the overall length of the DNA can be controlled to yield designer DNA that offers sequence-determined sites for further chemical adaptations, targeted small molecule binding studies, or sensing and sequencing applications.

Self-Priming Enzymatic Fabrication of Multiply Modified DNA.

We report a method for embedding cell-free protein synthesis reactions in macro-scale hydrogel materials without a free liquid phase. This paper focuses on methods of preparation for a variety of hydrogels and an investigation of the impact that the hydrogel material has on cell-free protein synthesis.

Cell-free protein synthesis in hydrogel materials.

Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of , and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

Article	Year
Duplex Healing of Selectively Thiolated Guanosine Mismatches through a Cd Chemical Stimulus. Chembiochem : a European journal of chemical biology\| PubMed ID: 29575493	2018
The Synthesis of Designer DNA. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29926443	2018
Self-Priming Enzymatic Fabrication of Multiply Modified DNA. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 29931815	2018
Cell-free protein synthesis in hydrogel materials. Chemical communications (Cambridge, England)\| PubMed ID: 32458833	2020
Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions. Computational and structural biotechnology journal\| PubMed ID: 35024094	2022

Colette J. Whitfield

Biography

JoVE Journal Publications

Other Publications