Biology subjects

Tyo, K.

Publications and source records attributed to Tyo, K..

3 recordsLinked to original sources

bioRxiv2024

Post-translation digital data encoding into the genomes of mammalian cell populations

High resolution cellular signal encoding is critical for better understanding of complex biological phenomena. DNA-based biosignal encoders alter genomic or plasmid DNA in a signal dependent manner. Current approaches involve the signal of interest affecting a DNA edit by interacting with a signal specific promoter which then results in expression of the effector molecule (DNA altering enzyme). Here, we present the proof of concept of a biosignal encoding system where the enzyme terminal deoxynucleotidyl transferase (TdT) acts as the effector molecule upon directly interacting with the signal of interest. A template independent DNA polymerase (DNAp), TdT incorporates nucleotides at the 3 OH ends of DNA substrate in a signal dependent manner. By employing CRISPR-Cas9 to create double stranded breaks in genomic DNA, we make 3OH ends available to act as substrate for TdT. We show that this system can successfully resolve and encode different concentrations of various biosignals into the genomic DNA of HEK-293T cells. Finally, we develop a simple encoding scheme associated with the tested biosignals and encode the message "HELLO WORLD" into the genomic DNA of HEK-293T cells at a population level with 91% accuracy. This work demonstrates a simple and engineerable system that can reliably store local biosignal information into the genomes of mammalian cell populations.

synthetic biology↗

bioRxiv2023

Meta-omic profiling reveals ubiquity of genes encoding for the high value nitrogen rich biopolymer cyanophycin in activated sludge microbiomes

Recovering nitrogen (N) from municipal wastewater is a promising approach to prevent nutrient pollution, reduce energy use, and transition towards a circular N bioeconomy, but remains a technologically challenging endeavor. Existing N recovery techniques are optimized for high-strength, low-volume wastewater. Therefore, developing methods to concentrate dilute N from mainstream wastewater will bridge the gap between existing technologies and practical implementation. The N-rich biopolymer cyanophycin is a promising candidate for N bioconcentration and recovery due to its solubility characteristics and potential for high levels of accumulation in a limited number of bacterial isolates. However, the cyanophycin synthesis pathway is poorly explored in natural and engineered microbiomes. In this study, we analyzed over 3700 publicly available metagenome assembled genomes (MAGs) and found that the cyanophycin synthesis gene cphA was ubiquitous across common activated sludge bacteria. Surprisingly, we found that cphA was present in all analyzed genomes of the common phosphorus accumulating organisms (PAO) Ca. Accumulibacter and Tetrasphaera, suggesting potential for simultaneous N and P bioconcentration in the same organisms. Using metatranscriptomic data, we also confirmed the expression of cphA in lab-scale bioreactors enriched with PAO. Our findings suggest that cyanophycin synthesis is a ubiquitous metabolic pathway in activated sludge microbiomes and therefore may have potential for integration in existing biological nutrient removal and recovery processes. We anticipate this work to be a starting point for future evaluations of combined N and P bioaccumulation, with the ultimate goal of advancing widespread adoption of nutrient recovery from municipal wastewater.

ecology↗

bioRxiv2021

Recording Temporal Signals with Minutes Resolution Using Enzymatic DNA Synthesis

Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with sub-hour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3 ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals like Co2+, Ca2+, Zn2+ concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co2+ concentration to within 1 minute over a 60-minute period. Finally, we engineer TdT to allosterically turn off in the presence of physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single step change in Ca2+ signal to within 1 minute over a 60-minute period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with minutes resolution.

synthetic biology↗