Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria

Yim, Sung Sun; Johns, Nathan I.; Noireaux, Vincent; Wang, Harris H.

doi:10.1021/acssynbio.0c00277

Cited by 30 publications

(56 citation statements)

References 31 publications

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“…However, because of Ku's different mechanism for LET protection, it was proven to be the most effective in lysates made from diverse bacteria. In B. subtilis and C. glutamicum extracts, Ku-protected LETs improved transcription 4.43-and 1.58-fold, respectively, compared to the no inhibitor control (Yim et al, 2020). This is notable improvement, considering GamS and Chi DNA addition to C. glutamicum CFEs actually reduced RNA yield slightly.…”

Section: Nuclease Inhibitionmentioning

confidence: 94%

“…Ku binds directly to dsDNA termini and is proven to reduce degradation by AdnAB, a helicase/nuclease in mycobacteria ( Sinha et al, 2009 ). Ku has been used recently in E. coli CFEs to protect LETs and improved transcription significantly, but those yields were only about a third of those transcribed when using GamS or Chi DNA oligos as inhibitors ( Yim et al, 2020 ). However, because of Ku’s different mechanism for LET protection, it was proven to be the most effective in lysates made from diverse bacteria.…”

Section: Approaches For Stabilizing Linear Dna In Cell-free Expression Systemsmentioning

confidence: 99%

“…Interestingly, Chi DNA sites have also been identified in other bacteria, such as B. subtilis and L. lactis , indicating that Chi DNA oligos could potentially be useful in extracts made from these bacteria ( Chedin and Kowalczykowski 2002 ; Marshall et al, 2017 ). Chi DNA inhibition has recently been tested in B. subtilis CFEs, and only improved LET yield about 2-fold ( Yim et al, 2020 ). At any rate, identifying which techniques are generalizable—and for those that are not generalizable, identifying alternatives or replacements as needed—will be important in moving this aspect of CFEs forward.…”

Section: The Future Of Linear Dna In Cell-free Expression Systemsmentioning

confidence: 99%

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Effective Use of Linear DNA in Cell-Free Expression Systems

McSweeney

Styczynski

2021

Front. Bioeng. Biotechnol.

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Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.

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Section: Nuclease Inhibitionmentioning

confidence: 94%

Section: Approaches For Stabilizing Linear Dna In Cell-free Expression Systemsmentioning

confidence: 99%

Section: The Future Of Linear Dna In Cell-free Expression Systemsmentioning

confidence: 99%

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Effective Use of Linear DNA in Cell-Free Expression Systems

McSweeney

Styczynski

2021

Front. Bioeng. Biotechnol.

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“…However, the protein expression yield of LDET-based cell-free systems with GamS is only one-third of plasmid-based cell-free systems ( Sun et al, 2014 ). There were also several studies focusing on DNA-binding proteins, such as Ku ( Yim et al, 2020 ) and scCro ( Zhu et al, 2020 ), to block the binding of native nucleases. There is still much room for improvement.…”

Section: Introductionmentioning

confidence: 99%

In silico Design of Linear DNA for Robust Cell-Free Gene Expression

Chen

2021

Front. Bioeng. Biotechnol.

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Cell-free gene expression systems with linear DNA expression templates (LDETs) have been widely applied in artificial cells, biochips, and high-throughput screening. However, due to the degradation caused by native nucleases in cell extracts, the transcription with linear DNA templates is weak, thereby resulting in low protein expression level, which greatly limits the development of cell-free systems using linear DNA templates. In this study, the protective sequences for stabilizing linear DNA and the transcribed mRNAs were rationally designed according to nucleases’ action mechanism, whose effectiveness was evaluated through computer simulation and cell-free gene expression. The cell-free experiment results indicated that, with the combined protection of designed sequence and GamS protein, the protein expression of LDET-based cell-free systems could reach the same level as plasmid-based cell-free systems. This study would potentially promote the development of the LDET-based cell-free gene expression system for broader applications.

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“…These include using the λ-phage GamS protein (Sitaraman et al 2004) or DNA containing Chi sites (Marshall et al 2017) to inhibit the RecBCD exonuclease complex. Protecting the linear DNA ends by DNA binding proteins has also been successful (Yim et al 2020;Norouzi, Panfilov, and Pardee 2021;Zhu et al 2020), as has the chemical modification of linear DNA ends (McSweeney and Styczynski 2021). All these methods still require supplementation of the extract with additional time-and cost-intensive components or modification of the template to achieve expression from linear DNA.…”

Section: Introductionmentioning

confidence: 99%

Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

Batista

Levrier

Soudier

et al. 2021

Preprint

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The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, we found that the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions. Finally, we illustrate the use of the δrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

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Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria

Cited by 30 publications

References 31 publications

Effective Use of Linear DNA in Cell-Free Expression Systems

Effective Use of Linear DNA in Cell-Free Expression Systems

In silico Design of Linear DNA for Robust Cell-Free Gene Expression

Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

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