Liu, J. D. | Search bioRxiv

bioRxiv2024

A Droplet Digital PCR Assay for Quantification of Bacteriophage Viral Vector Titer and Purity.

PurposeBacteriophage (phage) based vectors offer considerable promise as tools for tuning the microbiome with molecular and genetic precision. However, standardized methods to rigorously characterize phage vectors remain lacking. Here, we present an optimized digital droplet PCR (ddPCR)-based assay for quantifying both the purity and potency of phage vector preparations. MethodsWe utilized central composite design to develop a ddPCR assay capable of quantifying the number of phage vector capsids packed with the phage vector genome or packed with the transgenic DNA of interest. This assay targets 2 unique DNA barcodes, designed to be biologically inert and maximally orthogonal to existing DNA sequences. ResultsThrough stringent optimization, we were able to achieve assay conditions that enable a dynamic range of nearly 3 orders of magnitude and correct for systemic error in the assay. We then show that biological activity assays consistently underestimate transgene-packed vectors titers, leading to overestimation of true transduction efficiency, particularly when contamination by genome-packed vectors is high. We further demonstrate how this approach facilitates optimization of vector production conditions and substantially improves the precision and reproducibility of phage vector transduction. ConclusionCompared to assays of biological activity, this optimized ddPCR assay has improved accuracy and, through design of experiments optimization, high precision (CVs = 5.5 {+/-} 1.3% and 4.5 {+/-} 1.0% for the genome and transgene barcodes, respectively). This assay can be broadly adopted to characterize and quality control vector preparations for various applications.

microbiology↗

bioRxiv2022

Host translation machinery is not a barrier to phages that infect both CPR and non-CPR bacteria

Within human microbiomes, Gracilibacteria, Absconditabacteria, and Saccharibacteria, members of Candidate Phyla Radiation (CPR), are increasingly correlated with human oral health and disease. We profiled the diversity of CRISPR-Cas systems in the genomes of these bacteria and sought phages that are capable of infecting them by comparing their spacer inventories to large phage sequence databases. Gracilibacteria and Absconditabacteria recode the typical TGA stop codon to glycine and are infected by phages that share their hosts alternate genetic code. Unexpectedly, however, other predicted phages of Gracilibacteria and Absconditabacteria do not use an alternative genetic code. Some of these phages are predicted to infect both alternatively coded CPR bacteria and standard coded bacteria. These phages rely on other stop codons besides TGA, and thus should be capable of producing viable gene products in either bacterial host type. Interestingly, we predict that phages of Saccharibacteria can replicate in Actinobacteria, which have been shown to act as episymbiotic hosts for Saccharibacteria. Overall, the broad host range of some CPR phages may be advantageous for the production of these phages for microscopic characterization or use as therapy agents, given the current difficulty of CPR cultivation. Absconditabacteria phages and Gracilibacteria phages may have avoided acquisition of in-frame stop codons to increase the diversity of bacteria in which they can replicate.

microbiology↗