The endospore of Bacillus subtilis is formed intracellularly upon nutrient starvation and is encased by proteinaceous shells. The outermost layer, the crust, is a postulated glycoprotein layer that is composed of six proteins: CotV, W, X, Y, Z and CgeA. Despite some insight into protein interactions and the identification of players in glycosylation, a clear picture of its architecture is still missing. Here, we report a comprehensive mutational analysis that confirms CotZ as the anchor of the crust, while the crust structure is provided by CotV, CotX and CotY. CotY seems to be the major structural component, while CotV and CotX are polar and co-depend on each other and partially on CotW. CotW is independent of other crust proteins, instead depending on outer coat proteins, indicating a role at the interface of crust and coat. CgeA is coexpressed with putative glycosyltransferases (CgeB and CgeD) and implicated in crust glycosylation. In accordance, we provide evidence that CgeB, CgeCDE, SpsA-L, SpsM and SpsNOPQR (formerly YfnHGFED) contribute to the glycosylation state of the spore. The crust polysaccharide layer consists of functionally linked rhamnose-and galactose-related variants and could contain rare sugars. It may therefore protect the crust against biological degradation and scavenging. Protein interaction network and structure of the crustThe crust contains at least six different proteins: CotVWXYZ, which were long known as part of the insoluble fraction of the spore coat (Zhang et al.

The bottom-up approach in synthetic biology aims to create molecular ensembles that reproduce the organization and functions of living organisms and strives to integrate them in a modular and hierarchical fashion toward the basic unit of life—the cell—and beyond. This young field stands on the shoulders of fundamental research in molecular biology and biochemistry, next to synthetic chemistry, and, augmented by an engineering framework, has seen tremendous progress in recent years thanks to multiple technological and scientific advancements. In this timely review of the research over the past decade, we focus on three essential features of living cells: the ability to self-reproduce via recursive cycles of growth and division, the harnessing of energy to drive cellular processes, and the assembly of metabolic pathways. In addition, we cover the increasing efforts to establish multicellular systems via different communication strategies and critically evaluate the potential applications.

Upon starvation, the soil bacterium Bacillus subtilis forms an intracellular, metabolically inactive endospore. Its core contains the DNA, encased by three protein layers protecting it against a multitude of environmental threats. The outermost layer, the crust, harbors great potential as a protein-displaying platform: a gene of interest can be translationally fused to a crust protein gene, resulting in endospores displaying the desired protein on their surface. To unlock this potential in a standardized fashion, we designed a suite of 12 vectors (Sporovectors), based on the BioBrick cloning standard. With these vectors, proteins can easily be fused N- or C-terminally to the six crust proteins CotV, CotW, CotX, CotY, CotZ, and CgeA under the control of the strongest crust gene promoter P. All Sporovectors were evaluated with GFP and two different laccases. On the basis of our data, CotY and CotZ represent the best anchor proteins. But there are significant differences in activity and functional stability between the two tested laccases. Our vector suite is a powerful tool to generate and evaluate a vast variety of functionalized endospores. It allows quickly identifying the best anchor and fusion site for the protein of interest. Our findings demonstrate that the crust of B. subtilis endospores is an inexpensive and easy platform for displaying different proteins of interest.

Liposomes are used in synthetic biology as cell-like compartments and their microfluidic productiont hrough double emulsions allows for efficient encapsulationo fv arious components. However,r esidual oili nt he membrane remains ac riticalb ottleneck for creating pristine phospholipid bilayers. It has been discovered that osmotically driven shrinkingl eads to detachment of the oil drop. Separation inside am icrofluidic chip has been realizedt oa utomate the procedure, which allows for controlled continuous production of monodisperse liposomes.Giant unilamellar vesicles (GUVs)a re widely used as model membranes to study the biophysicalp roperties of phospholipid bilayers. [1][2][3] In parallel, they attract increasing attention as cell-like compartments in bottom-up synthetic biology,i n which the long-term goal is to build am inimal cell from scratch. [4][5][6][7][8] Upon selecting aG UV productionm ethod for synthetic biology,t he ability to encapsulate variousc omponents is essential. [2] Conventionalm ethods for the productiono fl iposomes comprise gentle hydration, [9,10] swelling on polymer cushions, [11,12] and electroformation. [13,14] These methods are not alwayso ptimal due to the low GUV yield in physiological buffer;p oor encapsulation efficiency; [2,15,16] and, in some cases, harsh conditions to which delicate biomolecules and smaller vesicles are exposed during preparation. [17] Thisi ssue has been addressed by the phase-transfer method, whichi sb ased on preformed water-in-oil (w/o) emulsion droplets crossing a second o/w interface. [18] In recent years, severalo ther,c oncep-tually similar,m ethods have been developed, with the aim of providing higher productivity andb etter control, namely,m icrofluidic jetting, [19] continuous droplet interface crossing encapsulation (cDICE), [20] microfluidic formation of droplet-stabilized vesicles, [21] and microfluidic production of w/o/w double emulsions. [22] The last approach appears to be the least experimentally demandinga nd multiple setups for double emulsion production have been proposed. Microfluidic chips made out of glass [17,22] or polydimethylsiloxane (PDMS), [23][24][25][26] and organic phases,s uch as octanol, [24] chloroform/hexane, [17] and oleic acid, [27] have been used to produce stable double emulsions, which have found attractive applications for synthetic biology, such as the encapsulationo fs maller vesicles, proteins, and DNA. [17,24,28] Another advantage of the double emulsionp rocedure is the virtual absence of losses, with respectt oe ncapsulated solutions,a nd therefore, it is suitable for valuable substratest hat are availablei nl ow quantities.In addition to efficient encapsulation, mimickingn ature requiresapristine bilayer,w hichw ould not compromise membrane-related phenomena, such as the folding of reconstituted membrane proteins.H owever,t he presence of residual oil is an inherentv ice of GUVs prepared from double emulsions, which necessitates removal of the organic phase. So far,afew approaches for solventr emoval have be...