A Selection for Intein Splicing inPhage Assisted Continuous Evolution
Name
hennes-ahennes-meng-eecs-2023-thesis.pdf
Description
Thesis PDF
Size
3.32 MB
Format
Adobe PDF
Checksum (MD5)
9e2316b12222017dea52867728d5d5b6
Author(s)
Hennes, Andrew
Advisor(s)
Wang, Xiao
Liu, David
Date Issued
June 2023
Publisher
Massachusetts Institute of Technology
Abstract
Developing tools for target manipulation of protein chemistry remains a longstanding goal in chemical biology. Trans-splicing inteins fulfill a crucial niche in this area by enabling post translational splicing of separate polypeptides, introduction of post translational modifications, and other chemistry on the amide backbone. This has motivated engineering and evolution of bespoke intein properties, including extein constraints, split site, kinetics, and orthogonality. Rational approaches are highly biased and preexisting directed evolution methods are laborious and often struggle with guaranteeing splicing dependance in a selection. To accelerate intein evolution we introduce a phage assisted continuous evolution (PACE) for intein properties. We show this selection is strictly splicing dependant, discriminates between intein splicing rates from one minute to several hours, discriminates between preferred over unpreferred extein contexts, supports propagation of phage in a multi-passage PANCE format, and circumvents recombination-driven phage cheating through a recombination-resistant helper strain. We anticipate this selection will enable facile, rapid evolution of inteins with bespoke properties.
MIT Department
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
Terms of Use
In Copyright - Educational Use Permitted
Copyright retained by author(s)
Persistent DSpace Link
