Biology subjects

Wannier, T. M.

Publications and source records attributed to Wannier, T. M..

6 recordsLinked to original sources

bioRxiv2025

Simulating a for-loop in the human genome: Design and evaluation of recombinase genetic programs that count to three.

Pluripotent cells specialize into numerous cell types by receiving external signals, making fate decisions, and executing differentiation functions - a paradigm similar to computer algorithms. While advances in biosensor design have enabled cells to respond to diverse stimuli, the ability to maintain a synthetic memory of the cells experiences that then informs its behaviors remains elusive. Here, we developed a system for cellular memory-driven behaviors by simulating a "for-loop" that counts to three using recombinase STepwise gene Expression Programs (STEPs). STEPs were genomically integrated into human cells, genotyped through targeted nanopore sequencing, and evaluated for function through changes in fluorescent reporter expression. While all STEPs were capable of heritable memory and sequential gene expression, the STEP design using tyrosine recombinases for successive excisions (TRex) significantly outperformed the others tested. We then used live cell imaging to track TRex cells as they incremented from count zero to three and observed the successive emergence of four cell states from an initially homogeneous population. The STEPs framework provides biological memory and conditional expression capabilities that, coupled with input mechanisms such as biosensors, can start to approach a programming language for biology.

synthetic biology↗

bioRxiv2022

helixCAM: A Platform for Programmable Cellular Assembly in Bacteria and Human Cells

Interactions between cells are indispensable for creating structure and signaling. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, CAMs are natively expressed by many cells and have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering novel CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. We built rationally designed helixCAM libraries and discovered novel high-performance helixCAM pairs. High-affinity helixCAMs were then used for multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning. The helixCAMs are an expandable platform for directing complex multicellular assemblies, and we foresee its utility in tissue engineering, immunology, and developmental biology.

synthetic biology↗

bioRxiv2020

High throughput functional variant screens via in-vivo production of single-stranded DNA

Tremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.

synthetic biology↗

bioRxiv2020

Improved bacterial recombineering by parallelized protein discovery

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, Multiplex Automated Genome Engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, highly efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call Serial Enrichment for Efficient Recombineering (SEER). By performing SEER in E. coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5 to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

microbiology↗

bioRxiv2017

Monomerization of Far-Red Fluorescent Proteins

Anthozoa class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red emitting variants ({lambda}em ~ 600 - 900 nm) sought for whole animal imaging because biological tissues are permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have usually produced dimmer and blue-shifted variants, as the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the vast majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the rapid monomerization of novel red-shifted oligomeric RFPs. Among the resultant variants, is mKelly1 (emission maximum: {lambda}em = 656 nm), which along with the recently reported mGarnet2 (1), forms a new class of bright, monomeric, far-red FPs.

biochemistry↗

bioRxiv2017

Long-term adaptive evolution of genomically recoded Escherichia coli

Efforts are underway to construct several recoded genomes anticipated to exhibit multi-virus resistance, enhanced non-standard amino acid (NSAA) incorporation, and capability for synthetic biocontainment. Though we succeeded in pioneering the first genomically recoded organism (Escherichia coli strain C321.{Delta}A), its fitness is far lower than that of its non-recoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for NSAA-linked applications requiring defined media such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.{Delta}A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceed the growth rates of both the ancestral C321.{Delta}A and non-recoded strains, permitting use of the recoded chassis in several new contexts. We use next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstruct individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occur only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared to application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.{Delta}A strain.\n\nSignificance StatementAfter demonstrating construction of an organism with an altered genetic code, we sought to evolve this organism for many generations to improve its fitness and learn what unique changes natural selection would bestow upon it. Although this organism initially had impaired fitness, we observed that adaptive laboratory evolution resulted in several selective mutations that corrected for insufficient translation termination and for unintended mutations that occurred when originally altering the genetic code. This work further bolsters our understanding of the pliability of the genetic code, it will help guide ongoing and future efforts seeking to recode genomes, and it results in a useful strain for non-standard amino acid incorporation in numerous contexts relevant for research and industry.

evolutionary biology↗