Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering.Along the way,combina...Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering.Along the way,combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits.In this report,we present an efficient evolutionary approach to build a range of regulatory control elements.The reported method allows for rapid construction of promoter,5'UTR,terminator and trans-activating RNA libraries.Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter.This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix.Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics.Specifically,both the promoter library and 50UTR library exhibits gene expression dynamics spanning across three order of magnitude.The terminator library and trans-activating RNA library displays relatively narrowed gene expression pattern.The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements.These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.展开更多
The corresponding author apologizes for listing Lynn Wong and Yun Jiao as co-authors of this manuscript.Triplicate experimental data,including fluorescence,cell density(OD)for the T7 promoter,T7 terminator,ribosome bi...The corresponding author apologizes for listing Lynn Wong and Yun Jiao as co-authors of this manuscript.Triplicate experimental data,including fluorescence,cell density(OD)for the T7 promoter,T7 terminator,ribosome binding sites(RBS)and trans-activating RNA libraries have been uploaded to Mendeley with the DOI number(https://doi.org/10.17632/skmnwht6dk.1).展开更多
基金The authors would like to acknowledge the Department of Chemical,Biochemical and Environmental Engineering,College of Engineering and Information Technology,Office of the Vice President for Research(gratn number 10145-1113-021-STRT7XUPMAIN)at University of Maryland Baltimore County for funding support.
文摘Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering.Along the way,combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits.In this report,we present an efficient evolutionary approach to build a range of regulatory control elements.The reported method allows for rapid construction of promoter,5'UTR,terminator and trans-activating RNA libraries.Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter.This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix.Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics.Specifically,both the promoter library and 50UTR library exhibits gene expression dynamics spanning across three order of magnitude.The terminator library and trans-activating RNA library displays relatively narrowed gene expression pattern.The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements.These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.
文摘The corresponding author apologizes for listing Lynn Wong and Yun Jiao as co-authors of this manuscript.Triplicate experimental data,including fluorescence,cell density(OD)for the T7 promoter,T7 terminator,ribosome binding sites(RBS)and trans-activating RNA libraries have been uploaded to Mendeley with the DOI number(https://doi.org/10.17632/skmnwht6dk.1).
