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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. Engineered Stop and Go T7 RNA Polymerases

   https://doi.org/10.1021/acssynbio.4c00627  

   Baumer, Zachary T; Newton, Matilda S; Löfstrand, Lina; Carpio_Paucar, Genesis Nicole; Farny, Natalie G; Whitehead, Timothy A (December 2024, ACS Synthetic Biology)

   Precise, stringent, post-translational activation of enzymes is essential for many synthetic biology applications. For example, even a few intracellular molecules of unregulated T7 RNA polymerase can result in growth cessation in a bacterium. We sought to mimic the properties of natural enzymes, where activity is regulated ubiquitously by endogenous metabolites. Here we demonstrate that full-length, single subunit T7-derived RNA polymerases (T7 RNAP) can be activated by physiologically relevant concentrations of indoles. We used rational design and directed evolution to identify T7 RNAP variants with minimal transcriptional activity in the absence of indole, and a 29-fold increase in activity with an EC50 of 344 μM. Indoles control T7-dependent gene expression exogenously, endogenously, and between cells. We also demonstrate indole-dependent bacteriophage viability and propagation in trans. Specificity of different indoles, T7 promoter specificities, and portability to different bacteria are shown. Our ligand activated RNA polymerases (LARPs) represent a new chemically inducible “stop and go” platform immediately deployable for novel synthetic biology applications, including for modulation of synthetic cocultures. 

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3. The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform

   https://doi.org/10.1093/synbio/ysab017  

   Garenne, David; Thompson, Seth; Brisson, Amaury; Khakimzhan, Aset; Noireaux, Vincent (August 2021, Synthetic Biology)

   null (Ed.)

   Abstract The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription–translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all-E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells. 

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4. Disease-associated inosine misincorporation into RNA hinders translation

   https://doi.org/10.1093/nar/gkac709  

   Schroader, Jacob H.; Jones, Lindsey A.; Meng, Ryan; Shorrock, Hannah K.; Richardson, Jared I.; Shaughnessy, Sharon M.; Lin, Qishan; Begley, Thomas J.; Berglund, J. Andrew; Fuchs, Gabriele; et al (August 2022, Nucleic Acids Research)

   Abstract Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis. 

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5. Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform

   https://doi.org/10.1093/synbio/ysac008  

   Sakai, Andrei; Deich, Christopher R; Nelissen, Frank H; Jonker, Aafke J; Bittencourt, Daniela M; Kempes, Christopher P; Wise, Kim S; Heus, Hans A; Huck, Wilhelm T; Adamala, Katarzyna P; et al (February 2022, Synthetic Biology)

   Abstract Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract 

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