Using Cell-Free Expression to Create Light-Activated Proteins In Situ in Droplet Interface Bilayer Networks
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Using Cell-Free Expression to Create Light-Activated Proteins In Situ in Droplet Interface Bilayer Networks Graham J. Taylor1 and Stephen A. Sarles*1 1 Department of Mechanical, Aerospace, and Biomedical Engineering, The University of Tennessee, Knoxville, TN 37920, U.S.A.
ABSTRACT Droplet interface bilayers (DIBs) are physical lipid bilayers that mimic real membranes in living cells, and they are formed quickly using droplets of water and lipids in oil (Fig. 1A). DIBs allow biomolecular sensing and direct detection of transmembrane proteins or peptides and small molecules such as drugs, anesthetics, or even ions. Cell-free expression systems allow in vitro protein synthesis using actual natural machinery extracted from organisms (Fig. 1B). Previous attempts to combine DIBs with cell-free extracts (CFE) encountered bilayer destabilization due to components in the expression system. This study evaluates incorporation of Promega’s T7 S30 High Yield (HY) Expression system with DIBs to pave the way for future in situ expression of light-activated bacteriorhodopsin (BR) and other complex transmembrane proteins in DIBs. A secondary output includes establishing a method for real-time monitoring and modeling of CF expression reactions using minimal volume. The ability to quantify CF output in such small volumes reduces cost per reaction from $20 to around $0.40, and synthesized protein levels reach tens to hundreds of micrograms per milliliter in less than 1 hour at 37°C. INTRODUCTION DIBs2-4 typically form highly resistive, impermeable seals between adjacent water droplets. Recent work suggests that DIBs are destabilized in the presence of cellfree extracts, either due to lipids or biomolecules in the lysate or polyethylene glycol supplements commonly added to expression systems.5-6 Given that the Promega HY kit used has not been tested with DIBs before, a primary objective is to characterize DIB formation with solutions combining the HY CFE and liposomes. To examine single molecule sensing capability of DIBs incorporating the HY extract, droplets are infused with alpha hemolysin (αHL), a well-known pore forming peptide.2, 7 Separately, Figure 1. A) A droplet interface bilayer forms spontaneously batch CF reaction products are used between two lipid-encased aqueous droplets in oil (Illustration by to form DIBs and CF reactions are S.A. Sarles1). B) Cell-free expression enables in vitro synthesis of performed in droplets. We explore proteins using only a plasmid/DNA containing a gene coding for the protein(s) of interest (POI).
the ability to both confirm and quantitatively monitor reaction output in real time in nanoliter sized droplets by using a gene for enhanced green fluorescent protein (eGFP). EXPERIMENTAL DETAILS CF Expression and DIB Formation. Reactions mixtures are prepared using the Promega T7 S30 HY Expression System per the manufacturer’s recommendations for all component ratios. The plasmid DNA used Figure 2. Typical DPhPC DIB formed between two has a pGOV4 backbone, the sequence for 300nL droplets of buffe
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