2026-09-19T00:58:24Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/1515302023-08-01T04:06:30Zcom_1721.1_7582com_1721.1_7581col_1721.1_131023
Wang, Xiao
Liu, David
Hennes, Andrew
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
2023-07-31T19:46:38Z
2023-07-31T19:46:38Z
2023-06
2023-06-06T16:35:05.217Z
https://hdl.handle.net/1721.1/151530
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.
M.Eng.
Massachusetts Institute of Technology
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https://rightsstatements.org/page/InC-EDU/1.0/
A Selection for Intein Splicing inPhage Assisted Continuous Evolution
Thesis
application/pdf
Master
Master of Engineering in Computer Science and Molecular Biology
Publication
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unknown
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<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.</Abstract>
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