Biology subjects

Gill, R. T.

Publications and source records attributed to Gill, R. T..

4 recordsLinked to original sources

bioRxiv2023

Engineered yeast cells simulating CD19+ cancers to control CAR T cell activation

Chimeric antigen receptor (CAR) T cells have become an established immunotherapy and show promising results for the treatment of hematological cancers. However, modulation of surface levels of the targeted antigen in cancer cells affects the quality and safety of CAR T cell therapy. Here we present the Synthetic Cellular Advanced Signal Adapter (SCASA) system, based on successful engineering of yeast to simulate cancer cells with tunable surface-antigen densities, as a tool for controlled activation of CAR T cell responses and assessment of antigen density effects. Specifically, we demonstrate I) controllable antigen-densities of CD19 on yeast using G protein-coupled receptors (GPCRs), II) a customizable system allowing choice of signal input and modular pathway engineering for precise fine-tuning of the output, III) synthetic cell-cell communication with CAR T cells and the application of CD19-displaying yeast in the characterization of CAR designs, and IV) more efficient and robust activational control of clinically-derived CAR T cells in comparison to the NALM6 cancer cell line. Based on this yeast-based antigen-presenting cell system, we envision efficient assessment of how varying antigen densities in cancer cells affect CAR T cell responses and ultimately support development of safer and better quality of personalized cancer therapies.

synthetic biology↗

bioRxiv2018

A versatile platform strain for high-fidelity multiplex genome editing

Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (1) a refactored {lambda}-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination efficiency, and (3) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

synthetic biology↗

bioRxiv2018

Isolation of Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to Fosmidomycin

Sequence to activity mapping technologies are rapidly developing, enabling the isolation of mutations that confer novel phenotypes. Here we used the CRISPR EnAbled Trackable genome Engineering (CREATE) technology to investigate the inhibition of the essential IspC gene in Escherichia coli. IspC gene product, Deoxyxylulose Phosphate Reductoisomerase (DXR), converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the DXP pathway. Since this pathway is shared with many pathogenic bacteria and protozoa and is missing in humans, it is an appealing target for inhibition. We created a full saturation library of 33 sites proximal to ligand binding and other sites and challenged it with the DXR-specific inhibitor, fosmidomycin. We identified several mutations that confer fosmidomycin resistance. All sites are highly conserved and also exist in pathogens including the malaria-inducing Plasmodium falciparum. These findings may have general implications on the isolation of resistance-conferring mutations and specifically, may affect the design of future generations of fosmidomycin-based drugs.\n\nSignificanceThe emergence of acquired drug resistance is a natural process that is likely to occur under most circumstances. Recently-developed technologies allow to map relative fitness contribution of multiple mutations in parallel. Such approaches may be used to predict which mutations are most likely to confer resistance, instead of waiting for them to evolve spontaneously. In this study, a rationally-designed IspC mutant library was generated genomically in E. coli. Mutants resistant to fosmidomycin, an antimalarial drug were identified, and most were in the highly conserved proline at position 274. These results may have implications on next-generation fosmidomycin drug design, and more broadly, this approach may be used for predicting mutational acquired resistance.

microbiology↗

bioRxiv2017

Refactoring the Genetic Code for Increased Evolvability

The standard genetic code is robust to mutations and base-pairing errors during transcription and translation. Point mutations are most likely to be synonymous or preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best performing mutant requires a replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyper-evolvable organism serving as an ideal platform for directed evolution experiments. We then conclude by evaluating potential applications for recoded organisms within the synthetic biology field.\n\nSignificance StatementThe conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene using common error-prone methods. Here we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transition to a larger pool of amino acids and with a greater degree of chemical differences, using a single nucleotide replacement within the codon, thus increasing evolvability both at the single gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation and fitness maximization.

bioengineering↗