Biology subjects

Deich, C.

Publications and source records attributed to Deich, C..

7 recordsLinked to original sources

bioRxiv2022

New Aequorea fluorescent proteins for cell-free bioengineering

Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms. O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/519681v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@3a3100org.highwire.dtl.DTLVardef@672233org.highwire.dtl.DTLVardef@f65f32org.highwire.dtl.DTLVardef@e40aaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection

Cell-free transcription-translation (TXTL) is an in vitro protein expression platform. In synthetic biology, TXTL is utilized for a variety of technologies, such as genetic circuit construction, metabolic pathway optimization, and building prototypes of synthetic cells. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still demanded. Here, we designed a novel method to control gene expression in TXTL, called a "silencing oligo." The silencing oligo is a short oligonucleotide that binds to the target mRNA. We demonstrated that addition of the silencing oligo inhibits eGFP expression in TXTL in a sequence-dependent manner. We investigated one of the silencing oligos inhibitory mechanisms and confirmed that silencing is associated with RNase H activity in bacterial TXTL reactions. We also engineered a transfection system that can be used in synthetic cells. We screened two dozen different commercially available transfection reagents to identify the one that works most robustly in our system. Finally, we combined the silencing oligo with the transfection technology, demonstrating that we can control the gene expression by transfecting silencing oligo-containing liposomes into the synthetic cells.

synthetic biology↗

bioRxiv2022

Parasites, infections and inoculation in synthetic minimal cells

Synthetic minimal cells provide a controllable and engineerable model for an increasing amount of biological processes. While much simpler than any live natural cell, synthetic cells offer a chassis for investigating the foundations of key biological processes. Here we show a synthetic cell system describing host cells interacting with parasites and infections of varying severity. We demonstrate how the host can be engineered to resist infection, we investigate the metabolic cost of carrying resistance, and we show a simple inoculation system that immunizes the host against pathogens. Our work expands the synthetic cell engineering toolbox by demonstrating host pathogen interactions and mechanisms for acquiring immunity. This brings synthetic cell systems one step closer to providing a comprehensive model of complex, natural life.

synthetic biology↗

bioRxiv2022

Expanding luciferase reporter systems for cell-free protein expression

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

synthetic biology↗

bioRxiv2022

Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems.

synthetic biology↗

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↗