Biology subjects

Isaacs, F. J.

Publications and source records attributed to Isaacs, F. J..

7 recordsLinked to original sources

bioRxiv2025

Engineered orthogonal translation systems from metagenomic libraries expand the genetic code

Genetic code expansion with non-canonical amino acids (ncAAs) opens new opportunities for the function and design of proteins by broadening their chemical repertoire. Unfortunately, ncAA incorporation is limited both by a small collection of orthogonal aminoacyl-tRNA synthetases (aaRSs) and tRNAs and by low-throughput methods to discover them. Here, we report the discovery, characterization, and engineering of a UGA suppressing orthogonal translation system mined from metagenomic data. We developed an integrated computational and experimental pipeline to profile the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS:tRNA pairs, and identify the AP1 TrpRS:tRNATrpUCA as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS:tRNATrpUCA is highly active in cell-free and cellular contexts. We then use Ochre, a genomically recoded Escherichia coli strain that lacks UAG and UGA codons, to engineer an AP1 TrpRS variant capable of 5-hydroxytryptophan incorporation at an open UGA codon. We anticipate that our strategy of integrating metagenomic bioprospecting with cell-free screening and cell-based engineering will accelerate the discovery and optimization of orthogonal translation systems for genetic code expansion.

synthetic biology↗

bioRxiv2025

Integrase-On-Demand: Bioprospecting integrases for targeted genomic insertion of genetic cargo

Integrases recognize defined sites within DNA and facilitate homologous recombination-independent transfer of genetic fragments. These innate capabilities render integrases as powerful biotechnology tools that mediate efficient site-specific integration of large genetic cargos. Given that genomes often lack the integration sites recognized by frequently utilized integrases, integrase technology has largely been restricted to genetic engineering of model organisms into which attB sites can be synthetically introduced. To enable single-step site-specific integrase-mediated genome editing in a broad spectrum of prokaryotes, we have devised the Integrase-On-Demand (IOD) method. IOD systematically identifies integrases, from bacteria and archaea, that can integrate into available attB sites in any target prokaryote. Computational results show that diverse bacteria generally have multiple potentially useable native attB/integrase pairs. We confirmed the functionality of predicted integrase and attB pairs for mediating site-specific genomic integration of heterologous DNA into the genomes of Pseudomonas putida S12 and KT2440 and Synechococcus elongatus UTEX 2973 and measured efficiency of integration using suicide constructs. By eliminating the requirement to introduce non-native attB sites into the target genome, IOD may, when suitable transformation methods exist, allow facile genome integration of large constructs in non-model and possibly non-culturable bacteria. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/676606v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1442a69org.highwire.dtl.DTLVardef@3c91b1org.highwire.dtl.DTLVardef@8131f4org.highwire.dtl.DTLVardef@1f6a206_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock

Hundreds of bacterial small open reading frames (sORFs) encoding microproteins of fewer than fifty amino acids have recently been identified. Biological functions have been ascribed to an increasing number of microproteins, some of which play roles in bacterial stress responses. In this work, we provide evidence that GndA, a frameshifted 36-amino acid microprotein encoded in an internal open reading frame overlapping the 6-phosphogluconate dehydrogenase (6PGD) coding sequence (CDS), functions in the response to heat shock. We demonstrate independent contributions of GndA and 6PGD to cell growth at high temperature. GndA is hydrophobic and associates with the pyruvate dehydrogenase complex and respiratory complex I (RCI). Consistent with a role in cellular homeostasis, GndA mutations alter the transcriptional responses to heat shock. These results demonstrate that GndA promotes bacterial cell growth at high temperature via a mechanism distinct from that of the 6PGD protein that it overlaps. GndA thus expands the paradigm of bacterial microproteins that function in cellular stress responses.

biochemistry↗

bioRxiv2022

Computational design and construction of an Escherichia coli strain engineered to produce a non-standard amino acid

Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para-aminophenylalanine (pAF) in Escherichia coli. First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism, and computationally identified metabolic interventions in the hosts native metabolism to improve pAF production. Next, we explored different ways of imposing these flux interventions experimentally and found that the upregulation of flux in chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers ([~]20 fold) while maintaining a reasonable pAF yield-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

systems biology↗

bioRxiv2020

DNA-Origami-Based Fluorescence Brightness Standards for Convenient and Fast Protein Counting in Live Cells

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in mammalian and bacterial cells, tagged with fluorescent proteins or organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

biophysics↗

bioRxiv2020

Advanced eMAGE for highly efficient combinatorial editing of a stable genome

Eukaryotic multiplex genome engineering (eMAGE) offers a powerful tool to generate precise combinatorial genome modifications in Saccharomyces cerevisiae. We optimize the design of synthetic oligonucleotides and enrichment of edited populations to increase editing frequencies up to 90%, reduce workflow time by 40%, and engineer a tunable mismatch repair system to lower the rate of spontaneous mutations [~]17-fold. These advances transiently evade genome maintenance to introduce multiple edits at high efficiencies in a stable genetic background, expanding utility of eMAGE in eukaryotic cells.

synthetic biology↗

bioRxiv2020

Tough Hydrogel-Based Biocontainment of Engineered Organisms for Continuous, Self-Powered Sensing and Computation

Genetically modified microorganisms (GMMs) can enable a wide range of important applications, including environmental sensing, precision therapeutics, and responsive materials. However, containment of GMMs to prevent environmental escape and satisfy regulatory requirements is a bottleneck for real-world use1-7. While biochemical strategies have been developed to restrict unwanted growth and replication of GMMs in the environment8-12, there is a need for deployable physical containment technologies to achieve redundant, multi-layered, and robust containment2. In addition, form factors that enable easy retrieval would be useful for environmental sensing. To address this challenge, we developed a hydrogel-based encapsulation system for GMMs that incorporates a biocompatible multilayer tough shell and an alginate-based core. This DEployable Physical COntainment Strategy (DEPCOS) allows no detectable GMM escape, bacteria to be protected against environmental insults including antibiotics and low pH, controllable lifespan, and easy retrieval of genetically recoded bacteria. To highlight the versatility of a DEPCOS, we demonstrate that robustly encapsulated cells can execute useful functions, including performing cell-cell communication with other encapsulated bacteria and sensing heavy metals in water samples from the Charles River. We envision that our multilayered physical and chemical containment strategy will facilitate the realization of a wide range of real-world applications for living biosensors.

synthetic biology↗