The freezing-thawing (F/T) method for fusing giant unilamellar vesicles (GUVs) can provide substrates, enzymes and membrane material simultaneously and repetitively, and is useful for constructing a recursive model of an artificial cell. However, enzymatic efficiency after F/T is reduced due to rupture of the GUVs and leakage of the inner solution during F/T. Previously, liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a negatively charged lipid, such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), showed lower rupture and leakage rates during F/T. In this study, we investigated the effect of POPG on the supply of components required for T7 RNA polymerase reactions via F/T by flow cytometry analysis. We found that the addition of POPG to liposome preparations reduced the efficiency of RNA synthesis. In addition, DNA was concentrated during F/T and RNA synthesis occurred after F/T in liposomes composed of POPC and POPG. Our results provide new insights for more efficient supply of substrates and enzymes by the F/T method, thereby increasing the utility of the F/T method for the construction of recursive bioreactors.