Living cells spatially organize biochemical reactions in compartments. Spatial organization in cell-free transcription and translation (TX-TL) reactions could increase synthesis rates and will be essential for the construction of artificial cells. Condensed arrangement of DNA has been shown to lead to increased protein yield in TX-TL reactions. We aim to examine whether we can create highly concentrated DNA compartments by liquid-liquid phase transitions. We aim to mimic the nucleolus, a membrane-less dense but liquid compartment in the nucleus of eukaryotic cells. To promote phase-transition in our in vitro system, we mix plasmids that carried arrays of the lac operator sequence (LacO) and its binding protein, lac repressor (LacI). In such mixtures, we observed small aggregates consisting of protein-crosslinked plasmids. The protein-DNA complex may potentially be a biomaterial to deliver and release DNA to desired destinations. Next, we plan to examine whether DNA-protein complexes promote the transcription efficiency of a weak promoter. Moreover, we plan to investigate the factors that influence the phase of the protein-DNA complex. This study will provide insights into the formation and function of membrane-less organelles and might result in a novel material for the construction of artificial cells.

Creating dense DNA phases with protein-crosslinked plasmids

255th National Meeting (2018)

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