Phage ELISA Kit

The ubiquitous human commensalEscherichia colihas been well investigated through its model representativeE. coliK-12. In this work, we initially characterizedE. coliFec10, a recently isolated human commensal strain of phylogroup A/sequence type ST10. Compared toE. coliK-12, the 4.88 Mbp Fec10 genome is characterized by distinct single-nucleotide polymorphisms and acquisition of genomic islands. In addition,E. coliFec10 possesses a 155.86 kbp IncY plasmid, a composite element based on phage P1. pFec10 harbours multiple cargo genes such as coding for a tetrathionate reductase and its corresponding regulatory two-component system. Among the cargo genes is also the Transmissible Locus of Protein Quality Control (TLPQC), which mediates tolerance to lethal temperatures in bacteria. The disaggregase ClpG(GI)of TLPQC constitutes a major determinant of the thermotolerance ofE. coliFec10. We confirmed stand-alone disaggregation activity, but observed distinct biochemical characteristics of ClpG(GI-Fec10)compared to the nearly identicalPseudomonas aeruginosaClpG(GI-SG17M.)Furthermore, we noted a unique contribution of ClpG(GI-Fec10)to the exquisite thermotolerance ofE. coliFec10, suggesting functional differences between both disaggregases in vivo. Detection of thermotolerance in 10% of human commensalE. coliisolates hints to the successful establishment of food-borne heat-resistant strains in the human gut.

Background The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA-despite being successful in E. coli and animal models-still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. Methods Previously, we have demonstrated the utility of our phage lambda-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a 'gene of interest'. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. Results The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of similar to 10kb and of a DUX4 reporter cassette of similar to 7kb in hESCs. Conclusion We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.