The Plesa Lab develops new technologies to characterize and engineer biological protein-based systems at large scales and applies these to a number of different research areas and applications. Our work is highly-interdisciplinary and touches on aspects from many fields including biochemistry, synthetic biology, molecular biology, microbiology, structural biology, genetics, bioinformatics, and quantitative biology.
We are developing new technologies to synthesize large DNA libraries. Biological systems are incredibly complex. Successfully understanding and engineering these systems on reasonable timescales requires large-scale testing of DNA-encoded hypotheses. To this end, we have developed technologies, such as DropSynth, to synthesize libraries of thousands of genes at low cost. DropSynth uses bead-based barcode hybridization to pull down the microarray-derived oligonucleotides necessary for a gene’s assembly, which are then processed and assembled in water-in-oil emulsions. This approach was previously used to assemble libraries of over >7,000 synthetic genes at a cost below $2 per gene (Plesa et al., Science 2018). We are actively developing new gene synthesis technologies with the goals of:
We are harnessing the immense natural diversity of receptor histidine kinases to build new biosensors. Bacteria have evolved a multitude of receptor histidine kinases to sense their outside world. These sensors, part of two-component systems, can detect many different small-molecule ligands and stimuli including nutrients, pH changes, quorum signaling molecules, metal ions, antibiotics, and other toxic compounds. Although hundreds of thousands of histidine kinases have been identified, with more being found regularly, very few of these have been characterized. Deciphering the function of members in this large protein family on a reasonable timescale will require the ability to experimentally characterize these systems at large scales. Our lab is building a platform to allow histidine kinase sensory domains to be characterized and engineered en masse by coupling them to a standardized output. This will allow us to both characterize their biological function and engineer novel receptors for ligands of interest against which natural sensors do not exist.
We are broadly interested in questions around the exploration of sequence space. Functional sequence space can be highly degenerate, containing many homologs with near identical functions despite having highly-divergent sequences as a result of evolutionary processes. Broad Mutational Scanning is a method to probe sequence-function relationships throughout an entire, functionally-related, protein family by assessing the impact of mutations around many different evolutionarily-related homologs simultaneously. We’re interested in addressing a number of open questions in this area:
The advent of next-generation sequencing has lead to an exponential increase in the amount of digital sequence information available in metagenomic databases. Synthetic metagenomics involves identifying sequences of interest from these databases, using gene synthesis to build the corresponding DNA molecules, and characterizing these en masse using functional assays. This approach leverages natural diversity to identify the optimal sequences for some desired functionality.
The emergence of antibiotic resistance has typically been studied in the context of genes from individual pathogenic organisms as opposed to a more community-wide approach, which would better reflect natural diversity of targets affected by broad-spectrum antibiotics. Understanding how resistance emerges given different evolutionary starting points will give us fundamental insights into its spread. We develop methods to tackle such fundamental questions at large scales by synthesizing large libraries of genes encoding antibiotic targets, introducing them into a model organism, and screening them (and their mutants) for resistance. Our current work focuses on dihydrofolate reductase (DHFR), a major antibiotic target in the folate acid biosynthesis pathway which is required for nucleic acid synthesis.