Biology subjects

Wong, D. A.

Publications and source records attributed to Wong, D. A..

5 recordsLinked to original sources

bioRxiv2025

Monitoring the stability of transgene expression in lettuce using the RUBY reporter.

Nearly four decades after the first transgenic lettuce was reported, constructs for stable transgene expression remain limited. Notably, the 35S promoter from the Cauliflower Mosaic Virus (35S), which drives strong expression of transgenes in several plant species, has often shown silencing and instability in lettuce. Other promoter/terminator combinations that are commonly used in plant expression vectors have not been extensively studied in lettuce. In this study, we evaluated three different expression constructs in two different horticultural types of lettuce using the non-invasive RUBY reporter, which allowed for the monitoring of transgene expression throughout the process of regeneration during tissue culture, throughout development of the primary transgenics, and in two subsequent sexual generations. The LsUBI promoter/terminator combination resulted in strong, uniform expression throughout regeneration, during growth of the primary transgenics, and in both subsequent generations. The AtUBI promoter/tRBCS combination showed slightly lower levels of expression and intermediate levels of silencing, while the 35S promoter/tHSP combination showed both initial strong expression and frequent silencing. Therefore, our data show that the LsUBI promoter/terminator combination provides strong, uniform expression that is unlikely to result in silencing and that the AtUBI promoter/tRBCS combination is an additional option for stable expression of transgenes in lettuce, especially if an intermediate expression level is desired.

plant biology↗

bioRxiv2025

A scalable cell-free manufacturing platform for two-step bioproduction of immunogenic conjugate vaccines

Rapid and decentralized vaccine production is essential to ensure global preparedness against emerging and re-emerging infectious diseases. Cell-free gene expression systems, which can be freeze-dried for long-term storage and re-activated for point-of-use synthesis, offer a promising solution to address this need. However, scalable cell-free production of conjugate vaccines--highly effective tools against bacterial infections--has been hindered by low yields and inefficient glycosylation. Here, we present an optimized, modular cell-free platform for the synthesis and purification of conjugate vaccines. By decoupling cell-free protein expression from in vitro glycosylation in a two-step approach, we achieve >85% glycosylation efficiency and up to [~]450 mg/L of glycoprotein. We apply this platform to manufacturing protein-polysaccharide conjugates composed of vaccine carrier proteins covalently modified with polysaccharide antigens from enterotoxigenic Escherichia coli O78 and Streptococcus pneumoniae serotype 4. Our workflow produced conjugate vaccine candidates in under 5 days with >87% product purity and low endotoxin levels suitable for preclinical evaluation. Immunization of mice with the pneumococcal conjugate vaccine induced a strong IgG response against the S. pneumoniae serotype 4 capsular polysaccharide, confirming the immunogenicity of the conjugate. We anticipate that this cell-free platform will advance efforts in decentralized manufacturing and rapid response to bacterial disease threats.

synthetic biology↗

bioRxiv2024

A cell-free workflow for detecting and characterizing RiPP recognition element-precursor peptide interactions

Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins and peptides. Current methods for studying and engineering PTM installing proteins often suffer from low-throughput experimental techniques. Here we describe a generalizable, in vitro workflow coupling cell-free protein synthesis (CFPS) with AlphaLISA for the rapid expression and testing of PTM installing proteins. We apply our workflow to two representative classes of peptide and protein therapeutics: ribosomally synthesized and post-translationally modified peptides (RiPPs) and conjugate vaccines. First, we demonstrate how our workflow can be used to characterize the binding activity of RiPP recognition elements, an important first step in RiPP biosynthesis, and be integrated into a biodiscovery pipeline for computationally predicted RiPP products. Then, we adapt our workflow to study and engineer oligosaccharyltransferases (OSTs) involved in conjugate vaccine production, enabling the identification of mutant OSTs and sites within a carrier protein that enable high efficiency production of conjugate vaccines. In total, we expect that our workflow will accelerate design-build-test cycles for engineering PTMs.

synthetic biology↗

bioRxiv2022

A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of travelers diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that significantly reduces mortality and morbidity associated with moderate to severe diarrhea among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic Escherichia coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS confers protection against ETEC infection in mice. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

synthetic biology↗

bioRxiv2022

A low-cost, thermostable, cell-free protein synthesis platform for on demand production of conjugate vaccines

Cell-free protein synthesis systems that can be lyophilized for long-term, nonrefrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability, as well as a lowcost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ~$0.50 per dose after storage at room temperature or 37 {degrees}C for up to 4 weeks and ~$1.00 per dose after storage at 50 {degrees}C for up to 4 weeks. We show that these conjugates generate bactericidal antibodies against enterotoxigenic E. coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our lowcost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.

synthetic biology↗