23:T4e5,The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as "gene compartments" for cell-free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane-free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of "cellular" physical environments on biochemical reactions in simplified cell-free systems.24:T470,Cell-free protein synthesis (CFPS) is a promising platform for protein engineering and synthetic biology. The storage of a CFPS system usually involves lyophilization, during which preventing the conformational damage of involved enzymes is critical to the activity. Herein, we report the protection role of magnesium ions on coupled transcription and translation in a lyophilized cell-free system. Mg²⁺ prevents the inactivation of the CFPS system from direct colyophilization of enzymes and substrates (nucleotides, and amino acids), and furthermore activates the CFPS system. We propose two-metal-ion regulation of Mg²⁺: Mg²⁺ (I) acts as an allosteric role for enzymes to prevent the conformational damage of enzymes from direct binding with substrates during lyophilization which locks up inactive enzyme-substrate complex; Mg²⁺ (II) consequently binds to enzymes to activate the CFPS system. Our work provides important implications for maximizing protein yields by using a cell-free system in protein engineering and understanding the functions of Mg²⁺ in biological systems.

#	Concept	H-Index	Publications	Citations
1	Natural Sciences	2	2	47
2	Engineering	1	1	28
3	Medicine	1	1	19
4	Biochemistry	1	1	19
5	Biomolecular Engineering	1	1	28

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