Cell-free microcompartmentalised transcription–translation for the prototyping of synthetic communication networks

Abstract

Graphical abstract

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Notably, genomic material withdrawal from chassis organism bestows circumstances in which desired synthesis reactions are achievable at high rates (Hodgman & Jewett, 2012). Thus, such features enable prevailing utilization of CFPS systems, which are also complementary to in vivo expression for various applications such as biocatalyst development (Rolf, Rosenthal, & Lütz, 2019), complex product fabrication (Chi, Wang, Li, Ren, & Huang, 2015), disease detection (Soltani, Davis, Ford, Nelson, & Bundy, 2018), glycoprotein synthesis (Jaroentomeechai et al, 2018), prototyping minimal cells (Yue, Zhu, & Kai, 2019), synthetic gene networks (Dubuc et al, 2019), as well as protein engineering (Hong, Kwon, & Jewett, 2014; Venkat, Chen, Gan, & Fan, 2019), among others (Table 1 and Figure 1).…”

“…Synthetic cells can afford compartments for the realization of CFPS, in the same way, TX‐TL machinery embedded inside synthetic cells can be used to build genetic circuits to mimic novel functions resembling living cells (Garamella, Garenne, & Noireaux, 2019). That is, synthetic biology traditionally utilizes genetic circuits to display complex spatiotemporal as well as collective behavior by establishing gene networks capable of establishing oscillations, synchronization, and combinatorial control of endogenous signaling pathways (Dubuc et al, 2019). Example of such complex behaviors, which leverage DNA‐based triggers, is replicated in proteinosomes (protein‐polymer vesicles; see Figure 2).…”

“…Notably, genomic material withdrawal from chassis organism bestows circumstances in which desired synthesis reactions are achievable at high rates . Thus, such features enable prevailing utilization of CFPS systems, which are also complementary to in vivo expression for various applications such as biocatalyst development (Rolf, Rosenthal, & Lütz, 2019), complex product fabrication (Chi, Wang, Li, Ren, & Huang, 2015), disease detection (Soltani, Davis, Ford, Nelson, & Bundy, 2018), glycoprotein synthesis (Jaroentomeechai et al, 2018), prototyping minimal cells (Yue, Zhu, & Kai, 2019), synthetic gene networks (Dubuc et al, 2019), as well as protein engineering (Hong, Kwon, & Jewett, 2014;Venkat, Chen, Gan, & Fan, Sheng, Lei, Yuan and Feng (2017) Functional CRISPR gene editing toolkit Marshall et al (2018) Pathway/network prototyping UDP-N-acetylglucosamine and UDP-GlcNAc pathway Zhou et al (2010) CRISPR-mediated gene activation and repression of the reporter and endogenous genes in mammalian cells Nakamura et al (2019) Protein engineering E. coli strain mutant for release factor 1 allows turning UAG termination codon into a sense codon for site-specific incorporation of nonnatural amino acids into proteins for selective conjugation with different biomolecules Adachi et al (2019) Incorporation of uAA AzF allows for site-specific mono and dipegylation of T4 Lysozyme Wilding et al (2018) Incorporating optimized nonnatural amino acids site-specifically, the feasibility of conjugating a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug by para-azidomethyl-L-phenylalanine (pAMF) to the tumor-specific, Her2-binding IgG Trastuzumab Zimmerman et al (2014) Biosensing Cheomogenic detection of estrogenic endocrine disruptors (hERb) in human blood and urine Salehi et al (2018) Glycoengineering Site-specific controlled glycosylation of proteins (glycoproteins) using E. coli extracts enriched for oligosaccharyltransferases and lipidlinked oligosaccharides Jaroentomeechai et al (2018) Site-directed incorporation of AzF, as well as ppropargyloxyphenylalanine from Sf21 insect cell, extracts for engineered O-glycosylation site of EPO Zemella et al (2018) Protein evolution: ribosome display Protein-tRNA-ribosome-mRNA complex for affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin Mattheakis, Bhatt and Dower (1994) Protein evolution: mRNA display Protein-puromycin-mRNA complex Roberts...…”

Establishing Communication Between Artificial Cells

“…Recent advances in CFPS technology, [37] gene synthesis and (droplet-based) microfluidics, [38] combined with rapid-prototyping techniques, [39] have rendered CFPS ap owerful platform for the programmable design of artificial cells and communication networks. [40] CFPS based artificial cells have also been suggested as ap romisingb ottom-up route towards minimal cells. [21,41] Optimization and integration of several sub-systems, [41c] possibly employing ae ukaryotic architecture, [42] will eventually lead to the realizationo fm ore complex synthetic cells.…”

Cell‐Free Protein Synthesis in Bifunctional Hyaluronan Microgels: A Strategy for In Situ Immobilization and Purification of His‐Tagged Proteins

“…The ongoing development of cell‐free protein synthesis (CFPS) platforms has significantly contributed to the understanding and mimicking of dynamic and functional aspects of living cells . In contrast to cell culture‐based approaches, the modularity of CFPS platforms provides remarkable opportunities regarding the synthesis of complex membrane proteins, cytotoxic proteins, or proteins containing unnatural amino acids .…”