Biology subjects

Koh, D. W.-S.

Publications and source records attributed to Koh, D. W.-S..

4 recordsLinked to original sources

bioRxiv2022

Augmenting the Signal Peptide of the Ag43 Autotransporter for the improved heterologous display of sfGFP using Fluorescence-Activated Cell Sorting (FACs)-assisted natural selection.

Protein display, secretion and export in prokaryotes are essential for utilizing microbial systems as engineered living materials for medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that increased surface display from 1.4 to 3 folds as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the protein was displayed extracellularly, we trypsinised the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging on epPCR and single-cell sorting to rapidly augment bacterial surface display, a method that could be applied to other bacterial proteins.

synthetic biology↗

bioRxiv2021

Augmenting Whole Recombinant Antibody Production in HEK293E Cells: Optimizing Transfection and Culture Parameters

BackgroundOptimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations. MethodsInvestigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements. ResultsUsing 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 g respectively in a cost-effective manner. With the addition of peptone, {kappa} light chain increased by ~4-fold to 1032 g while whole antibody increased to a lesser extent by ~2.5-fold to 51 g, with benefits potentially for antibodies limited by their light chains in production. ConclusionsOur optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells. Statement of SignificanceRecombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~4-fold light chain alone production increase to 1032 g and a 2.5-fold whole antibody production increase to 51 g from 2 mL transfections.

molecular biology↗

bioRxiv2020

Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero-waste circular economy

Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to natural biodegradation. Recent research showed that meal-worms (Tenebrio molitor) and superworms (Zophobas morio) are naturally able to consume PS as a carbon food source and degrade them without observable toxic effects. In this study, we explored the effects of possible food waste contamination and use of worm frass as potential plant fertilizers. We found that small amounts of sucrose and bran increased PS consumption and that the worm frass alone could support dragon fruit cacti (Hylocereus undatus) growth, with superworm frass in particular, supporting better growth and rooting than mealworm frass and control media over a fortnight. As known fish and poultry feed, these finding present worms as a natural solution to simultaneously tackle both the global plastic problem and urban farming issue in a zero-waste sustainable bioremediation cycle.

ecology↗

bioRxiv2019

Predicting mutations in HIV-1 Gag: Insights from in silico and an in vitro BSL2 platform on thermostability and allosteric effects

While drug resistant mutations in HIV-1 is largely credited to its error prone HIV-1 RT, host proteins such as deaminases may also play a role generating mutations. Many HIV-1 RT mutational in vitro studies utilize reporter genes (LacZ) as template, leaving out the possible contribution of HIV codon usage and gene-specific effects. To address this gap, we studied HIV-1 RT mutation rates and bias on its own Gag, protease, and RT p66 genes in an in-vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease and RT p66 at 4.71 x 10-5, 6.03 x 10-5, and 7.09 x 10-5 mutations/bp respectively. Gag and p66 genes showed a large number of A to G hypermutations likely due to cellular adenosine deaminases. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 x 10-4 mutations/bp) and that A to G mutations occurred in regions reminiscent of ADAR neighbour preferences. Mutational free energies by the A to G mutations revealed an avoidance of destabilizing effects with the natural p66 gene codon usage providing barriers to ADAR effects. Our study demonstrates the importance of studying mutation emergence in HIV genes to understand how fast drug resistance can emerge, sometimes with contributions of host deaminases, providing transferable applications to how new viral diseases and drug resistances can emerge.

microbiology↗