The recently developed yeast three-hybrid system is a powerful tool for analyzing RNA-protein interactions in vivo. However, large numbers of false positives are frequently met due to bait RNA-independent activation o...The recently developed yeast three-hybrid system is a powerful tool for analyzing RNA-protein interactions in vivo. However, large numbers of false positives are frequently met due to bait RNA-independent activation of the reporter gene in the library screening using this system. In this report, we coupled the colony color assay with the 5FOA (5-fluoroorotic acid) negative selection in the library screening, and found that this coupled method effectively eliminated bait RNA-independent false positives and hence greatly improved library screening efficiency. We used this method successfully in isolation of cDNA of an RNA-binding protein that might play important roles in certain cellular process. This improvement will facilitate the use of the yeast three-hybrid system in analyzing RNA-protein interaction.展开更多
Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces i...Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable.In this study,we developed a screening system for targeted gene knockout using a uracil auxotrophic host(ΔpyrF)resistant to the highly toxic uracil analog of 5-fluoroorotic acid(5-FOA)converted by PyrF,and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase.The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications.Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutantΔpyrF at the targeted locus.Double-crossover recombinants were generated,from which the pyrF gene,plasmid backbone,and targeted gene were excised through homologous recombination exchange.These recombinants were rapidly screened by the counterselection agent,5-FOA.We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene,which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018.This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.展开更多
基金the State "863" High-Tech R&D Project (Grant No. 863-102-11-03-04).
文摘The recently developed yeast three-hybrid system is a powerful tool for analyzing RNA-protein interactions in vivo. However, large numbers of false positives are frequently met due to bait RNA-independent activation of the reporter gene in the library screening using this system. In this report, we coupled the colony color assay with the 5FOA (5-fluoroorotic acid) negative selection in the library screening, and found that this coupled method effectively eliminated bait RNA-independent false positives and hence greatly improved library screening efficiency. We used this method successfully in isolation of cDNA of an RNA-binding protein that might play important roles in certain cellular process. This improvement will facilitate the use of the yeast three-hybrid system in analyzing RNA-protein interaction.
基金This work is supported by the Natural Science Foundation of Hebei Province(No.C2019209399)Tangshan Science and Technology Project(No.20130208b)+1 种基金the Science and Technology Program of Hebei(No.18222916)the Research Fund for Top Discipline Construction of North China University of Science and Technology(No.18060720),China.
文摘Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable.In this study,we developed a screening system for targeted gene knockout using a uracil auxotrophic host(ΔpyrF)resistant to the highly toxic uracil analog of 5-fluoroorotic acid(5-FOA)converted by PyrF,and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase.The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications.Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutantΔpyrF at the targeted locus.Double-crossover recombinants were generated,from which the pyrF gene,plasmid backbone,and targeted gene were excised through homologous recombination exchange.These recombinants were rapidly screened by the counterselection agent,5-FOA.We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene,which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018.This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.
