A Selection for Intein Splicing inPhage Assisted Continuous Evolution

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.

Name thesis:degree_name

Master

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2023

Author dc:creator

• Hennes, Andrew

Advisors dc:contributor.advisor

• Wang, Xiao
• Liu, David

Statement dc:rights

• In Copyright - Educational Use Permitted
• Copyright retained by author(s)

Licence dc:rights.uri

• https://rightsstatements.org/page/InC-EDU/1.0/

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/151530

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/151530