Light-induced protein expression within droplet interface bilayers

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Even though there has been substantial progress in the preparation of membrane proteins, these proteins are usually cumbersome to handle since they tend to aggregate in aqueous solution. Expression of these proteins in the vicinity of the target membrane provides an alternative as incorporation of the protein into the membrane can prevent its aggregation. Droplet interface bilayers (DIBs) are a useful membrane system since they provide a stable compartment, enclose only small volumes and have been shown to be useful for applications such as rapid screening of ion channels. Ideally, protein expression in DIBs would be temporally controlled. In this study, the implementation of a light-inducible in vitro transcription/translation (IVTT) system within droplets is pursued. Triggering the expression by light allows delivery of the IVTT system to droplets without transcription occurring and storage of DIBs ready to express membrane proteins on demand. Two strategies will be applied: The first one takes advantage of the surfactant azoTAB which was shown to facilitate DNA condensation in its trans conformation. Light-induced isomerization to the cis state leads to the release of DNA, permitting transcription. The second strategy aims to control the activity of T7 RNA polymerase, an enzyme crucial for transcription in various IVTT systems. Introducing a caged amino acid at a critical position in the polymerase will lead to an inactive enzyme which can be uncaged and therefore activated by light of a distinct wavelength.