Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule, the DNA, must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the singlecell level in droplet-based microfluidics. Here, we compare three different expression systems for L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia. We developed three expression vectors to produce and localize L-asparaginase (L-ASNase) in E. coli either in the cytoplasm, on the surface of the inner membrane (display), or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.
With this personal account we show how the Integrative Chemistry, when combining the sol-gel process and concentrated emulsions, allows to trigger inorganic, hybrid or living materials when dedicated toward heterogeneous catalysis applications. In here we focus on 3D-macrocellular monolithic foams bearing hierarchical porosities and applications thereof toward heterogeneous catalysis where both activities and mass transport are enhanced. We thereby first depict the general background of emulsions, focusing on concentrated ones, acting as soft templates for the design of solid (HIPE) foams, HIPE being the acronym for High Internal Phase Emulsions while encompassing both sol-gel and polymer chemistry. Secondly we extend this approach toward the design of inorganic cellular materials labeled Si(HIPE) and hybrid organic-inorganic foams, labeled Organo-Si(HIPE), where heterogeneous catalysis applications are addressed considering acidic, metallic, enzymatic and bacterial-based modified Si-HIPE. Along, we will show how the fluid hydrodynamic within the macrocellular foams is offering advanced "out of the box" heterogeneous catalytic capabilities.
Microorganisms are highly efficient biocatalysts. Yet making use of their capabilities for chemical transformations requiring synergistic interactions between different microbes is challenging as the competition for resources might reduce the diversity and ultimately disrupt the synergies. Here, a new method is proposed for constructing microbial consortia for the integration of multistep transformations. Bacteria are successively grown and trapped within semipermeable inorganic foams produced as millimeter‐sized beads. The beads function as efficient living biocatalysts is demonstrated. These living heterogeneous biocatalysts are manipulated to perform cycles of biochemical reactions and furthermore assembled to perform preprogrammed sequences of reactions. This new family of living advanced biocatalysts should find applications in a wide range of basic research and industrial systems where complex tasks have to be performed by controlled consortia of microorganisms.
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