Multiplexed gene synthesis in emulsions for exploring protein functional landscapes

Area of Science:

• Synthetic biology
• Molecular biology
• Genomics

Background:

• Efficient construction and functional characterization of DNA sequences are crucial for biological research.
• Current methods for large-scale gene synthesis and functional testing can be costly and time-consuming.

Purpose of the Study:

• To introduce DropSynth, a scalable, low-cost method for pooled, multiplexed synthesis of gene-length DNA constructs.
• To demonstrate the utility of DropSynth for large-scale functional genomics by analyzing gene homolog function.

Main Methods:

• DropSynth utilizes barcoded beads to capture necessary oligonucleotides for gene assembly.
• Oligonucleotides are processed and assembled into gene constructs within water-in-oil emulsions.
• Multiplexed functional assays were performed to test the complementation activity of synthetic genes in *Escherichia coli*.

Main Results:

• Successfully synthesized over 7000 defined, gene-length DNA constructs using DropSynth.
• Identified phylogenetically diverse homologs of essential *Escherichia coli* genes.
• Determined core functional motifs and reasons for functional incompatibility among gene homologs through multiplexed testing.

Conclusions:

• DropSynth provides a scalable and cost-effective platform for synthetic gene construction.
• Coupling DropSynth with multiplexed functional assays enables unprecedented exploration of sequence-function relationships.
• This approach accelerates the understanding of gene function and evolution.