Man-made cell-like compartments for molecular evolution

Abstract

Cellular compartmentalization is vital for the evolution of all living organisms. Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype. We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions. These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts. A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product. In other compartments, substrates attached to genes that do not encode catalysts remain unmodified. Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product. We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system. A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion. Genes encoding HaeIII methyltransferase were selected from a 10(7)-fold excess of genes encoding another enzyme.

Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4‐helix bundle proteins

“…53 The availability of our superfamily of unevolved proteins with low levels of promiscuous activity will enable further exploration into several aspects of molecular evolution. The collection can be combined with highthroughput screens such as phage display, 54 mRNA display, 55 and in vitro compartmentalization, 56 or with genetic selections in vivo 57 to isolate de novo proteins with high levels of unique (non-promiscuous) activity. Ultimately, the isolation of biologically active proteins from a designed library of de novo sequences would represent a significant advance in synthetic biology.…”

Dynamics of microfluidic droplets

“…3 The idea of performing chemical or biochemical reactions in droplets had already been explored, before the microfluidics era, through the use of emulsions in order to ''compartmentalize'' reactions inside many small parallel volumes. 4,5 The introduction of microfluidics tools again acts to facilitate the production and manipulation of these compartments.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…The ease of determination and regulation of droplet content and the large number of droplets (>10 10 per milliliter emulsion) makes IVC a suitable ultra-high-throughput screening technology. In the first application of this technology, genes encoding methyltransferases were selected from a 10 7 -fold excess of genes encoding another enzymes (Tawfik and Griffiths 1998). Application of IVC together with FACS in metagenomics, as an advantage over traditional functional screening, has been reviewed in detail by ).…”