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1. Akaby—Cell-free protein expression system for linear templates

   https://doi.org/10.1371/journal.pone.0266272  

   Sato, Wakana; Sharon, Judee; Deich, Christopher; Gaut, Nathaniel; Cash, Brock; Engelhart, Aaron E.; Adamala, Katarzyna P. (April 2022, PLOS ONE)

   Perez-Fernandez, Jorge (Ed.)

   Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based onEscherichia coli(E.coli). Endogenous nucleases inE.colicell-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 inE.coli, with the RecB subunit possessing the actual nuclease activity. We created aRecBknockout of anE.colistrain 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 sourceE.colistrain, enabling the creation of TXTL systems with other custom modifications. 

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2. Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1

   https://doi.org/10.1016/j.mimet.2024.106920  

   McFarlane, John A; Garenne, David; Noireaux, Vincent; Bowden, Steven D (May 2024, Journal of Microbiological Methods)

   NA (Ed.)

   Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system. 

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3. Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

   https://doi.org/10.3791/66595  

   Loi, Kyle J; Moghimianavval, Hossein; Liu, Allen P (March 2024, Journal of Visualized Experiments)

   Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While expression of soluble proteins is usually successful in common CFE systems, reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful co-translational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells. 

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4. Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

   https://doi.org/10.1038/s41467-021-22329-3  

   Hershewe, Jasmine M.; Warfel, Katherine F.; Iyer, Shaelyn M.; Peruzzi, Justin A.; Sullivan, Claretta J.; Roth, Eric W.; DeLisa, Matthew P.; Kamat, Neha P.; Jewett, Michael C. (December 2021, Nature Communications)

   null (Ed.)

   Abstract Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli- based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N- linked and O -linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities. 

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5. Enhancer activation via TCP and HD-ZIP and repression by Dof transcription factors mediate giant cell-specific expression

   https://doi.org/10.1093/plcell/koad054  

   Hong, Lilan; Rusnak, Byron; Ko, Clint S; Xu, Shouling; He, Xi; Qiu, Dengying; Kang, S Earl; Pruneda-Paz, Jose L; Roeder, Adrienne H (February 2023, The Plant Cell)

   Abstract Proper cell-type identity relies on highly coordinated regulation of gene expression. Regulatory elements such as enhancers can produce cell type-specific expression patterns, but the mechanisms underlying specificity are not well understood. We previously identified an enhancer region capable of driving specific expression in giant cells, which are large, highly endoreduplicated cells in the Arabidopsis thaliana sepal epidermis. In this study, we use the giant cell enhancer as a model to understand the regulatory logic that promotes cell type-specific expression. Our dissection of the enhancer revealed that giant cell specificity is mediated primarily through the combination of two activators and one repressor. HD-ZIP and TCP transcription factors are involved in the activation of expression throughout the epidermis. High expression of HD-ZIP transcription factor genes in giant cells promoted higher expression driven by the enhancer in giant cells. Dof transcription factors repressed the activity of the enhancer such that only giant cells maintained enhancer activity. Thus, our data are consistent with a conceptual model whereby cell type-specific expression emerges from the combined activities of three transcription factor families activating and repressing expression in epidermal cells. 

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