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, 2 (4), e1600056
eCollection

Light-activated Communication in Synthetic Tissues

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Light-activated Communication in Synthetic Tissues

Michael J Booth et al. Sci Adv.

Abstract

We have previously used three-dimensional (3D) printing to prepare tissue-like materials in which picoliter aqueous compartments are separated by lipid bilayers. These printed droplets are elaborated into synthetic cells by using a tightly regulated in vitro transcription/translation system. A light-activated DNA promoter has been developed that can be used to turn on the expression of any gene within the synthetic cells. We used light activation to express protein pores in 3D-printed patterns within synthetic tissues. The pores are incorporated into specific bilayer interfaces and thereby mediate rapid, directional electrical communication between subsets of cells. Accordingly, we have developed a functional mimic of neuronal transmission that can be controlled in a precise way.

Figures

Fig. 1
Fig. 1. Construction and evaluation of a light-activated promoter.
(A) T7 RNA polymerase is blocked from binding to the LA-T7 promoter due to the presence of multiple monovalent streptavidins, bound to the DNA through biotinylated PC linkers. Following UV light cleavage of the linkers, T7 RNA polymerase can transcribe the downstream gene. (B) LA-T7 promoter sequence. Pink-colored thymines are replaced with amino-C6-dT modifications and the primary amines of the nucleobase coupled to the PC biotin group. (C) LA-DNA encoding for mVenus is only expressed upon UV irradiation. There is no significant difference between expression from the LA-DNA (+UV) and expression from the amine-only DNA construct. a.u., arbitary unit.
Fig. 2
Fig. 2. Light-activated expression of LA-mVenus in synthetic cells and synthetic tissues.
(A) Schematic of a synthetic cell that will express mVenus protein upon light activation, where a single bilayer connects it to a neighboring synthetic cell. IVTT, in vitro transcription and translation. (B) Synthetic cells containing LA-mVenus DNA express mVenus protein (yellow) upon light activation. (C) Fluorescence intensity line profiles from (B). (D) Schematic of 3D-printed synthetic tissues containing hundreds of synthetic cells. A single lipid bilayer, as shown in (A), connects each cell with its neighbor. (E) Synthetic tissues containing LA-mVenus DNA express mVenus protein (yellow) upon light activation. (F) Fluorescence intensity line profiles from (E).
Fig. 3
Fig. 3. Light-activated expression from LA–αHL-GFP DNA in a pair of synthetic cells forming a DIB.
(A) Schematic of the synthetic cell pair. One cell contains LA–αHL–green fluorescent protein (GFP) DNA, the other contains no DNA. The αHL-GFP fusion pore protein will localize to the bilayer when expressed. (B) When LA–αHL-GFP DNA is activated in a synthetic cell neighboring another containing no DNA, the αHL-GFP fusion membrane pore protein locates to the bilayer. No expression is observed without light activation. (C) Fluorescence intensity line profiles from (B). (D) Rotated 3D projection of a z stack of αHL-GFP DNA, as expressed in (B), demonstrates that αHL-GFP becomes located throughout the flat interface bilayer.
Fig. 4
Fig. 4. Light-activated electrical signal between synthetic cells.
(A) Schematic of the synthetic cell pair. One cell contains LA-αHL DNA, the other contains no DNA. Both droplets have electrodes inserted within them to apply a potential and measure the ionic current. Below is an image of the experimental setup. (B) A current is detected only following the expression of αHL after light activation. (C) Voltage protocol used in (B).
Fig. 5
Fig. 5. Recording of electrical communication in 3D-printed synthetic tissues mediated by LA-αHL DNA.
(A) Schematic of 3D-printed synthetic tissue containing LA-αHL DNA, which expresses the αHL membrane pore upon light activation. (B) Electrical recordings from a 3D-printed network demonstrate that a current through the synthetic tissues is only detected upon light activation. The voltage protocol used to detect αHL insertion into bilayer is also shown.
Fig. 6
Fig. 6. Light-activated patterned expression of mVenus from LA-mVenus DNA in a 3D-printed synthetic tissue.
(A) Schematic showing a printed tissue with droplets containing LA-mVenus DNA (yellow) and droplets containing no DNA (gray). (B) After light activation, the mVenus-containing droplets become visible (yellow fluorescence).
Fig. 7
Fig. 7. Electrical recordings from an L-shaped pathway formed by expression from LA-αHL DNA in a 3D-printed synthetic tissue.
(A) Schematic of the printed tissue containing droplets with LA-αHL DNA (red) printed with droplets containing no DNA (clear region within black frame). Numbers represent sides of the cuboid where electrodes were placed to detect the conductive pathway. (B to F) Electrical recordings detect a current when the electrodes are at positions 1 and 2 (B), based on the voltage protocol in (B), but not when one or both of the electrodes are positioned off the pathway sides 1 and 3 (C), 1 and 4 (D), 1 and 5 (E), or 3 and 5 (F).

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