Synthetic cells are engineered vesicles that can mimic one or more salient features of life. These features include directed localization, sense‐and‐respond behavior, gene expression, metabolism, and high stability. In nanomedicine, many of these features are desirable capabilities of drug delivery vehicles but are difficult to engineer. In this focus article, we discuss where synthetic cells offer unique advantages over nanoparticle and living cell therapies. We review progress in the engineering of the above life‐like behaviors and how they are deployed in nanomedicine. Finally, we assess key challenges synthetic cells face before being deployed as drugs and suggest ways to overcome these challenges. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology‐Inspired Nanomaterials > Lipid‐Based Structures

Global dissemination of New Delhi metallo-β-lactamase (NDM)-producing bacteria has become a major health threat. However, there are few reports regarding the identification and characterisation of NDM-producing bacteria from West Africa, including Ghana. An Escherichia coli strain with resistance to meropenem was isolated from the Tamale Teaching Hospital in Ghana. Its identification and determination of antibiotic susceptibility profile were carried out using commercial systems. The antibiotic resistance mechanism was analysed by phenotypic detection kits, PCR, and DNA sequencing. Conjugation experiments, S1 nuclease pulsed field gel electrophoresis, and Southern blotting were performed. Finally, the NDM-1-harbouring plasmid was characterised using next-generation sequencing and phylogenetic analysis. The meropenem-resistant Escherichia coli strain EC2189 harboured blaNDM-1 and belonged to sequence type 410. blaNDM-1 was located on the IncHI type transferrable plasmid p2189-NDM (248,807 bp long), which co-carried multiple resistance genes, such as blaCTX-M-15, aadA1, aac(6')-Ib, sul3, dfrA12, and cmlA1. p2189-NDM phylogenetically differed from previously identified blaNDM-1-positive IncHI type plasmids. A truncated Tn125 containing blaNDM-1 was bracketed by an ISSm-1-like insertion sequence upstream and by a site-specific integrase downstream. To the best of our knowledge, we have, for the first time identified and molecularly characterised an NDM-1-producing Enterobacteriaceae strain in Ghana with blaNDM-1 that had a novel genetic structure. Our findings indicate a possibility of NDM-1 dissemination in Ghana and underscore the need for constant monitoring of carbapenemase-producing bacteria.

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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.

Diarrhoeagenic Escherichia coli (DEC), an important agent of infectious diarrhoea, is constantly evolving, making it necessary for its periodic monitoring. Unfortunately, DEC genotypes in Ghana remain uncharacterised. We focused on characterising the molecular serotypes, virulence factors, multilocus sequence types, and the phylogenetic relatedness among different DEC pathotypes recovered from stool samples of paediatric patients with diarrhoea symptoms from the Western region of Ghana. We detected all five common DEC pathotypes, with majority being enterotoxigenic E. coli (ETEC) isolates harbouring the heat-labile enterotoxin gene. The DEC strains exhibited a diverse serotypic identity with novel and other outbreak strains. Sequence type (ST)38, ST316, and ST1722 were the most prevalent STs, and clonal complex (CC)10 was the most common CC. A close evolutionary distance was observed among most of the isolates. Coli surface antigen 6 was the most prevalent (44%, n = 11) ETEC-specific colonisation factor. Nearly all of the isolates harboured lpfA, and the frequencies of other virulence genes, such as pap and cnf1, were 7.9% and 18.4%, respectively. This study provides insights into the important and novel genotypes circulating in the Western region of Ghana that should be monitored for public health.

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.

Extra-intestinal pathogenic Escherichia coli (ExPEC) is one of the world’s leading causes of bloodstream infections with high mortality. Sequence type 410 (ST410) is an emerging ExPEC clone resistant to a wide range of antibiotics. In this study, we investigated the epidemiology of 21 ST410 E. coli isolates from two Ghanaian hospitals. We also investigated the isolates within a global context to provide further insight into the dissemination of this highly pathogenic clone. A phylogenetic tree of the 21 isolate genomes, along with 102 others from global collection, was constructed representing the ensuing clades and sub-clades of the ST: A/H53, B2/H24R, B3/H24Rx, and B4/H24RxC. The carbapenem-resistant sub-clade B4/H24RxC is reported to have emerged in the early 2000s when ST410 acquired an IncX3 plasmid carrying a blaOXA–181 carbapenemase gene, and a second carbapenemase gene, blaNDM–5, on a conserved IncFII plasmid in 2014. We identified, in this study, one blaOXA–181–carrying isolate belonging to B4/H24RxC sub-lineage and one carrying blaNDM–1 belonging to sub-lineage B3/H24Rx. The blaOXA–181 gene was found on a 51kb IncX3 plasmid; pEc1079_3. The majority (12/21) of our Ghanaian isolates were clustered with international strains described by previous authors as closely related strains to B4/H24RxC. Six others were clustered among the ESBL-associated sub-lineage B3/H24Rx and three with the globally disseminated sub-lineage B4/H24RxC. The results show that this highly pathogenic clone is disseminated in Ghana and, given its ability to transmit between hosts, it poses a serious threat and should be monitored closely.

Background Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Results Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. Conclusions 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.