Project description:Directed evolution (DE) is a process of mutation and iterative artificial selection to breed biomolecules with new or improved activity. DE platforms are primarily prokaryotic or yeast-based, and stable highly mutagenic mammalian systems have been challenging to establish and apply. To this end, we developed PROTein Evolution Using Selection (PROTEUS), a new platform that uses chimeric virus-like vesicles (VLVs) to enable extended mammalian DE campaigns without loss of system integrity. This platform, consisting of a minimal modified Semliki Forest virus genome controlling expression of the Indiana vesiculovirus G coat protein, is stable and can generate sufficient diversity for DE in mammalian systems. Using PROTEUS, we altered the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation. PROTEUS is also compatible with intracellular nanobody evolution, and we use it to design a novel DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is a robust, efficient, and stable platform to direct evolution of biomolecules within mammalian cells.
Project description:Directed evolution (DE) is a process of mutation and iterative artificial selection to breed biomolecules with new or improved activity. DE platforms are primarily prokaryotic or yeast-based, and stable highly mutagenic mammalian systems have been challenging to establish and apply. To this end, we developed PROTein Evolution Using Selection (PROTEUS), a new platform that uses chimeric virus-like vesicles (VLVs) to enable extended mammalian DE campaigns without loss of system integrity. This platform, consisting of a minimal modified Semliki Forest virus genome controlling expression of the Indiana vesiculovirus G coat protein, is stable and can generate sufficient diversity for DE in mammalian systems. Using PROTEUS, we altered the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation. PROTEUS is also compatible with intracellular nanobody evolution, and we use it to design a novel DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is a robust, efficient, and stable platform to direct evolution of biomolecules within mammalian cells.
Project description:Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (UTIs) and urolithiasis. The transcriptional regulator MrpJ inversely modulates two critical aspects of P. mirabilis UTI progression: fimbria-mediated attachment to the urinary tract, and flagella-mediated motility. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was used for the first time in a CAUTI pathogen to probe for in vivo direct targets of MrpJ. ChIP-seq revealed 81 78 direct MrpJ targets, including genes for motility, fimbriae and a type VI secretion system (T6SS), and the putative MrpJ binding sequence ACnCnnnnnnnGnGT.
Project description:Microorganisms with high selenite-tolerant and efficient reduction ability of selenite have seldom been reported. In this study, a highly selenite-resistant strain (up to 500 mM), isolated from lateritic red soil, was identified as Proteus penneri LAB-1. Remarkably, isolate LAB-1 reduced nearly 2 mM of selenite within 18 h with the production of selenium nanoparticles (SeNPs) at the beginning of the exponential phase. Moreover, in vitro selenite reduction activities of strain LAB-1 were detected in the membrane protein fraction with or without NADPH/NADH as electron donors. Strain LAB-1 transported selenite to the membrane via nitrate transport protein. The selenite was reduced to SeNPs through the glutathione pathway and the catalysis of nitrate reductase, and the glutathione pathway played the decisive role. P. penneri LAB-1 could be a potential candidate for the selenite bioremediation and SeNPs synthesis.