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. 2022 Jul 5;13:3879. doi: 10.1038/s41467-022-31467-1

Fig. 2. Microbial swarmbot supported the growth of various microbes and produced versatile proteins and small molecules.

Fig. 2

a The growth of engineered E. coli was supported in microbial swarmbot (MSB). MG1655 cells (constitutively producing mCherry) were encapsulated in chitosan capsules and cultured in the SC + LB medium at 30 °C. Images were taken at 24 h. Scale bar = 200 μm. b The growth of engineered S. cerevisiae was supported in MSB. BY4742 cells (expressing GFP under the TDH3 promoter) were encapsulated in chitosan capsules and cultured in the SC + LB medium at 30 °C. Images were taken at 24 h. Scale bar = 200 μm. c MSB(E. coli) produced the model protein through the programmed self-lysis of the cells. We used a density-dependent circuit (ePop) to program the autonomous lysis of the E. coli. MSB(E. coli) (BL21(DE3) carrying the ePop circuit and the GFP-fused polypeptides expression circuit) were cultured for 24 h in M9 medium. The supernatant was collected for His-tagged resins purification. The resultant protein was verified by SDS-PAGE. Experiments were repeated independently more than three times with similar results. d MSB(P. pastoris) produced a therapeutic protein through the engineered secretion. We created the circuit by fusing the sequence of recombinant human growth hormone (rh-GH) with α-factor signal for secretory expression. The circuit (driven by the AOXI promoter) was then linearized and integrated into the genome of P. pastoris X-33 by electroporation. We generated the MSB(P. pastoris) and cultured them in the BMMY medium for 96 h. The supernatant was then collected, purified by His-tagged resins and verified by SDS-PAGE. Experiments were repeated independently more than three times with similar results. e MSB(S. cerevisiae) produced cannabigerolic acid (CBGA) by feeding olivetolic acid (OA). MSB (S. cerevisiae) were cultured in 1 mM olivetolic acid and assayed for CBGA production using liquid chromatography. The decrease in the OA peak and increase in the CBGA peak indicated the production of CBGA by consuming OA. Source data are provided as a Source Data file.