Laboratory evolution has generated many proteins and nucleic acids with desired properties, but a single round of directed evolution typically requires days or longer with frequent intervention by the researcher. Here we describe a new platform that enables the continuous directed evolution, in principle, of any gene-encoded molecule that can be linked to the production of a protein in E. coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle. The activity of interest is linked to phage replication through production of pIII, a protein required for phage infection. Because phage begin releasing progeny ∼10 minutes after infection and mutagenesis occurs continuously, dozens of cycles of phage replication, mutation, and selection can occur in a single day without human intervention. Using PACE, we continuously evolved T7 RNA polymerase to recognize a distinct DNA promoter and to initiate transcripts with nucleotides other than GG. Each of the three evolved activities emerged in less than one week of continuous evolution. PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions in molecular evolution.
|Advisor:||Liu, David R.|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 74/01(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Evolution and Development, Biochemistry|
|Keywords:||Continuous evolution, Directed evolution, Filamentous phages|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be