Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression

Mullin, Alissa C.; Slouka, Taylor; Oza, Javin P.

doi:10.1007/978-1-0716-1998-8_2

Cited by 5 publications

(5 citation statements)

References 23 publications

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“…Cell extracts were prepared using the E. coli strain BL21*DE3, based on the CFAI method. ,, 1L of LB broth was inoculated with BL21*DE3 cells and was incubated in a shaking incubator at 200 rpm and 30 °C overnight. All centrifugation steps were performed using a Beckman Coulter Avanti J-E centrifuge.…”

Section: Methodsmentioning

confidence: 99%

Development of Solid-State Storage for Cell-Free Expression Systems

et al. 2023

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The fragility of biological systems during storage, transport, and utilization necessitates reliable cold-chain infrastructure and limits the potential of biotechnological applications. In order to unlock the broad applications of existing and emerging biological technologies, we report the development of a novel solid-state storage platform for complex biologics. The resulting solid-state biologics (SSB) platform meets four key requirements: facile rehydration of solid materials, activation of biochemical activity, ability to support complex downstream applications and functionalities, and compatibility for deployment in a variety of reaction formats and environments. As a model system of biochemical complexity, we utilized crudeEscherichia colicell extracts that retain active cellular metabolism and support robust levels of in vitro transcription and translation. We demonstrate broad versatility and utility of SSB through proof-of-concepts for on-demand in vitro biomanufacturing of proteins at a milliliter scale, the activation of downstream CRISPR activity, as well as deployment on paper-based devices. SSBs unlock a breadth of applications in biomanufacturing, discovery, diagnostics, and education in resource-limited environments on Earth and in space.

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Section: Methodsmentioning

confidence: 99%

Development of Solid-State Storage for Cell-Free Expression Systems

et al. 2023

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“…Kanamycin (30 μg/ml) was added to the media for cultures containing pET30-derived vectors and pJL1-sfGFP. The BL21(DE3) strain was used to prepare CFAI-based CFPS extracts as previously described ( Levine et al, 2020 ; Smith et al, 2021 ; Mullin et al, 2022 ). The CFAI media auto-induces T7 RNAP expression during cell growth, and cells are harvested at high ODs.…”

Section: Methodsmentioning

confidence: 99%

“…Barriers to access have reduced significantly as CFPS systems have become commercially available in the form of kits derived from lysates of a variety of chassis organisms, as well as reconstituted systems ( Shimizu et al, 2005 ). For this study, the New England Biolab’s NEBExpress and NEB PURExpress kits were used alongside our in-house E. coli lysate-based CFAI system to assess the effects that distinct vector elements have on protein synthesis ( Levine et al, 2020 ; Smith et al, 2021 ; Mullin et al, 2022 ). Both the NEBExpress and CFAI systems utilize crude E. coli extracts.…”

Section: Introductionmentioning

confidence: 99%

Characterizing and Improving pET Vectors for Cell-free Expression

Jew

Smith

et al. 2022

Front. Bioeng. Biotechnol.

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Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.

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“…Not avoidable [88] Avoidable [89,90] Not avoidable [72,73] Avoidable [91] Dunkelmann et al Codon availability for ncAAs c) 3 (stop codons) [43,68,86] Up to 44 [92] Hong et al Optimal reaction time (based on the E. coli system) 1-24 h [93] 2 h [94] Mullin et al Integration of display technique mRNA e) display [95] mRNA e) display [96] Ribosome display [97] noncanonical monomers would be very challenging. However, several recent studies have shown that ribosomes are, in fact, amenable to synthesizing new types of polymers bearing noncanonical chemical substrates.…”

Section: Production Yieldsmentioning

confidence: 99%

Programmable Synthesis of Biobased Materials Using Cell‐Free Systems

et al. 2022

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Motivated by the intricate mechanisms underlying biomolecule syntheses in cells that chemistry is currently unable to mimic, researchers have harnessed biological systems for manufacturing novel materials. Cell‐free systems (CFSs) utilizing the bioactivity of transcriptional and translational machineries in vitro are excellent tools that allow supplementation of exogenous materials for production of innovative materials beyond the capability of natural biological systems. Herein, recent studies that have advanced the ability to expand the scope of biobased materials using CFS are summarized and approaches enabling the production of high‐value materials, prototyping of genetic parts and modules, and biofunctionalization are discussed. By extending the reach of chemical and enzymatic reactions complementary to cellular materials, CFSs provide new opportunities at the interface of materials science and synthetic biology.

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Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression

Cited by 5 publications

References 23 publications

Development of Solid-State Storage for Cell-Free Expression Systems

Development of Solid-State Storage for Cell-Free Expression Systems

Characterizing and Improving pET Vectors for Cell-free Expression

Programmable Synthesis of Biobased Materials Using Cell‐Free Systems

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