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Effect of adding mycoplasma lysate and CaCl2 to E. coli CFE. (A) Expression of eGFP decreases by adding increasing amounts of Syn3A or Mcap lysate from 0% (100 vol% E. coli lysate) to 100% (100 vol% mycoplasma lysate). (B) Addition of CaCl2 to ∼15 mM restored eGFP expression in a 1:1 E. coli:Syn3A CFE reaction. An E. coli codon-optimized plasmid under the control of a T7 promoter was used for E. coli CFE reaction of eGFP.
