Cell-like platforms are being studied intensively for their application in synthetic biology to mimic aspects of life in an artificial environment. Here, micrometer-sized, bifunctional microgels are used as an experimental platform to investigate the interplay of cell-free protein synthesis (CFPS) and in situ protein accumulation inside the microgel volume. In detail, microgels made of hyaluronic acid (HA) are first modified with different amounts of nitrilotriacetic acid (NTA) moieties to characterize the capability and maximum capacity of binding His-tag modified GFP. CFPS is optimized for the system used here, particularly when using a linear DNA template. Afterward, HA-microgels are functionalized with the linear DNA template and Ni