Journal: bioRxiv

Article Title: Dairy practices select for thermostable endolysins in phages

doi: 10.1101/2023.09.27.559745

Figure Lengend Snippet: A phage CaBM mutant with a D219A substitution was created by changing the arginine present in the second position of the domain to a cysteine. The phage mutant was then serially transferred with its host for a total of thirty transfers, each conducted with a final volume of 10 ml and a hundred-fold dilution rate. n=3. B) Throughout each of these transfers, phage-infected cells were exposed to a range of thermal conditions, including temperatures of 37°C and 46°C, as well as a 15-minute heat shock at 60°C. Additionally, infected cells were also exposed to thermal conditions that mimic those encountered during the production of pressed cooked cheese. C) After the thirty transfers, targeted sequencing of the CaBM at the population level was performed with Illumina MiSeq to measure the SNP frequency (C to A reversion) under the different incubation conditions. The detection limit was set based on the initial SNP frequency found in the phage population before the transfers.

Article Snippet: Libraries for high-throughput sequencing of PCR products were prepared following the “16S Metagenomic Sequencing Library Preparation” protocol by Illumina, using primers with extensions containing the adapters and the MiSeq Reagent Kit v2 (CaBM_D219A_Fw and CaBM_D219A_Rv, Supplementary Table 1).

Techniques: Mutagenesis, Infection, Sequencing, Incubation