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

Fig. 3. 34-strain microbial swarmbot consortium enables the assembly of core machinery of PURE.

Fig. 3

a The core machinery of protein synthesis in E. coli requires 34 enzymes. In the first step of protein synthesis, 22 aminoacyl-tRNA synthetases (Phe-tRNA synthetase and Gly-tRNA synthetase have two subunits for each) catalyze the aminoacylation reaction by covalently linking an amino acid to its cognate tRNA. Methionyl-tRNA transformylase attaches a formyl group to the free amino group of methionyl-tRNA. The translation factors responsible for initiation, elongation and termination are three initiation factors (IF1, IF2, and IF3), four elongation factors (EF-G, EF-Tu, EF-Ts, and EF-4), three release factors (RF1, RF2, and RF3) and ribosome recycling factor (RRF). b Individually cultured 34 different MSBs produced each of the 34 enzymes. A circuit (ePop) programmed the density-dependent autonomous lysis of the E. coli. The cells were trapped inside the polymeric scaffold, while the proteins were transported to the exterior of the MSB. The supernatant was collected and purified for examination. SDS-PAGE showed the expression of 34 proteins by individually cultured MSBs. Experiments were repeated independently more than three times with similar results. c Mass spectrometry results suggested the presence of all 34 elements, as purified from the supernatant of the microbial swarmbot consortium (MSBC). We co-cultured the MSBC consisting of 34 MSBs, collected the supernatant and purified it with His-tagged resins. The purified elements were subject to the mass spectrometry quantification. The y axis is in arbitrary units. d Reconstitution of the reaction using the MSBC produced 34 enzymes led to the RFP expression. Addition of the template DNA (+template) drove the expression of RFP in the reaction. When the reaction is not supplemented with template DNA (−template), there was no signal detected. The y axis is in log(arbitrary unit). Error bars =  Standard Deviation (n = 3 biologically independent samples). Source data are provided as a Source Data file.