Synthetic cells with self-activating optogenetic proteins communicate with natural cells

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells’ capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.


Plasmids and hosts
The DNA sequences of all the proteins used in this study are listed in supplementary table 1. E. coli DH5α and TOP10 strains were used for cloning and plasmid purification.
DNA sequences of the engineered plasmids were confirmed by sequencing. A plasmid expressing Renilla luciferase (Rluc) under the T7 promoter was obtained from the S30-T7 high yield protein expression system kit, purchased from Promega (USA). Gaussia luciferase (Gluc)-expressing plasmid was generously provided Prof. James Swartz (department of chemical engineering, Stanford university). Plasmids expressing DsbC (Plasmid #38152), EL222 (Plasmid #113108), iLID (Plasmid #60408), sspB-Nano (Plasmid #60409), and pBLind RFP (Plasmid #113109) were purchased from Addgene (USA). PCR was used to add a C-terminal his tag to the DsbC protein and insert it to a pET28a vector. C-terminal his tag was also added to the EL222 sequence that was isolated and inserted into a pET28a vector. Hifi DNA assembly (NEB, USA) was used to produce Gluc-EL222-his and his-Gluc-iLID sequences, each inserted to a pet28a vector (deposited in Addgene, IDs #172097 and #172096 respectively). mRFP1 sequence was inserted between the MBP and sspB-Nano reading frames to generate a his-MBP-RFP-sspB-Nano vector. A plasmid expressing Rluc under the pBLind promoter was produced by replacing the RFP sequence in the original vector with the Rluc sequence from the Rluc expressing plasmid. A complete list of the primers used for plasmid design is available in Supplementary Table 1.

Protein expression and purification
BL21(DE3) E. coli (NEB) were used for the expression of DsbC-his, EL222-his, iLID and his-maltose binding protein (MBP)-mRFP1-sspB-Nano. Expression of Gluc-EL222-his and his-Gluc-iLID was performed in SHuffle T7 E. coli. A 5 ml Luria-broth starter culture for each protein was incubated overnight at 37 ºC and 250 rpm with the compatible antibiotics (ampicillin at 100 μg ml -1 or kanamycin at 25 μg ml -1 ). The starter was then transferred to 500 ml of Terrific-broth supplemented with antibiotics in the same concentration and grown at 37 ºC and 250 rpm to optical density (OD) of 0.5, when they were induced with 500 μM of Isopropyl β-D-1-thiogalactopyranoside (IPTG). DsbC-his and EL222-his were incubated at 37 ºC and 250 rpm following S5 induction until reaching an OD of ~4. His-iLID, his-MBP-mRFP1-sspB-Nano, Gluc-EL222-his and his-Gluc-iLID were grown at 16 ºC and 250 rpm until reaching similar OD values. Cells were harvested by centrifugation at 7,000 x g for 10 minutes at 4 °C and kept at -20 °C until the next step.
For protein purification, the pellet was resuspended in PBS (in the case of DsbC-his and EL222-his), 50 mM Tris, 300 mM NaCl, pH 7.4 (in the case of his-iLID and his-MBP-RFP-sspB-Nano) or 300 mM NaCl, 50 mM phosphate buffer, pH 8.0 (in the case of his-Gluc-iLID and Gluc-EL222-his). The cells were fractionated by two passes through an emulsiFlex-C3 high pressure homogenizer (Avestin, Germany) and the lysate was spun down two times for 15 minutes at 20,000 x g. The supernatant was passed through an AKTA purifier chromatography system (Cytiva, USA) using a HisTrap HP 5 ml column and eluted with elution buffer with similar composition to the loading buffer supplemented with 500 mM imidazole. The protein containing fractions were dialyzed in a 12-14 kD membrane (Spectrum Laboratories, USA) against their original resuspension buffer.
To remove the his-MBP domain from the mRFP1-SspB-Nano protein, the eluted MBP-RFP-sspB proteins were cut with TEV protease (NEB) using a digestion site between the MBP and the RPF sections. 300 μg of his-MBP-RFP-sspB-Nano were diluted to a total reaction volume of 880 μl. 20 μl of TEV Protease Reaction Buffer (10X) and 100 μl of TEV Protease were added, and incubated at 4°C overnight. 10 reactions samples were pooled together and passed through a Ni Sepharose 6 Fast Flow histidine-tagged protein purification resin (Cytiva). The flow-through containing the RFP-SspB-Nano protein, was collected and concentrated using Amicon ultra 15 kDa (Merck, USA). The proteins were dialyzed overnight in PBS.  Data is represented as the mean ± standard deviation (n=3 independent samples).

Reagent final concentration
Nested two-tailed t-test adjusted P value; *p=0.048. Data is represented as the mean ± standard deviation (n=2 independent samples). UPW denotes ultra-pure water. Figure 13. SDS-Page gel with coomassie blue staining of the his-MBP-mRFP-sspB-Nano after TEV restriction, before and after purification of mRFP-sspB-Nano. Gel electrophoresis analysis of the purification process was performed once.