Design,Patterned Assembly,and Application Of Escherichia Coli Biofilm CsgA Amyloid Materials

The development of biomacromolecule-based coating and patternable materials with environmental tolerance,functional diversity,and fabrication flexibility would drive many technological innovations in biophotonics,bioelectronics,biosensors,biomedicine,tissue engineering,and other domains.However,existing biomacromolecule-based coating and patternable materials can hardly integrate the aforementioned attributes.As such,their relevant applications are often limited.The protein matrix of Escherichia coli biofilm is enriched with an amyloid protein called Csg A,which can self-assemble into nanofibers.The assembled nanofibers have many attractive properties: First,Csg A has characteristic ?-sheet structures,which in turn endow the nanofibers with excellent mechanical properties and environmental tolerance.Secondly,Csg A proteins can be functionalized with diverse and programmable functions using a modular genetic strategy.Csg A nanofibers therefore represent a new type of highly promising functional material that combines environmental tolerance and functional diversity.Based on the above considerations,this dissertation therefore aims to rationally integrate genetic engineering,protein engineering,soft lithography and corresponding material fabrication and characterization methods to develop a series of Csg A protein molecular materials.Furthermore,the dissertation also aims to develop new nanofiber coating technology and nanofiber patterning strategy by using functional selfassembling Csg A proteins as building blocks.To fulfill these goals,corresponding designs and experiments were divided into three different yet connected sessions: a)Engineering functional Csg A proteins via a modular genetic strategy;b)Fabrication and applications of functional Csg A nanofiber coatings,and 3)Patterned assembly of functional Csg A nanofibers into hierarchically ordered structures and devices,and their relevant applications.In the first session,a modular genetic strategy was used to engineer Csg A proteins containing multiple functional domains.The self-assembly mechanisms,ranging from macro-,micro-,to molecular level,and their programmable functions were fully probed and explored.Such knowledge provides theoretical guidance for rational design of Csg A amyloid proteins and serves as the basis for the subsequent development of nanofiber coating and patterning techniques.In nature,Escherichia coli cells attach to diverse substrates through adhesive Csg A amyloid nanofibers.Inspired by such natural phenomenon,the second part of this dissertation was therefore devoted to explore the development and application of Csg A nanofibers as conformal and universal coating materials with extremely strong chemical/thermal stability.Accordingly,the following proof-of-concept devices were constructed and demonstrated with innovative functions including pressure sensor,touch switch,multi-enzyme bioconversion systems,microfluidic sensors,MOF mediated-synthesis,and lithium battery separator modification.Compared with exsiting coating materials include self-assembled monolayers,silane,polyelectrolytes,polydopamine,and polyphenol,Csg A nanofiber coatings exhibit several advantages including facile fabrication,substrate independence,structural stability,and environmental tolerance.The third part of this dissertation mainly focused on patterning assembly of functional Csg A amyloids into diverse hierachical architectures and devices.To such ends,hexafluoroisopropanol(HFIP)and methanol were applied for controlling the selfassembly of Csg A protein in situ: HFIP could effectively disrupt extensive interactions amongst ?-sheets and prevent the self-assembly of Csg A until the monomers were properly in place.Following their deposition(here in hierarchical defined micro-and even nana-scale patterns)using standard fabrication methods like soft lithography,Csg A monomers were then exposed to methanol vapor,which would trigger their reassembly into ?-sheet nanofiber structures in situ.This idea for a generic amyloid HFIP ink,when merged with a top-down fabrication,along with a bottom-up,methanol-assisted curing step,ultimately unlocked the problem of how to fabricate amyloid materials into patterned structures and devices with highly precise and complex spatial configurations.It bears emphasis that intense tests with a large number of harsh agents(hexane,acetone,isopropanol,etc.)and extreme conditions(high temperature,tape-peeling,water jetting,etc.)demonstrate the ultra-stability of Csg A patterns.Finally,various patterned structures and devices based on the patterned assembly of Csg A nanofibers were constructed with multiple exciting proof-of-concept applications,including bioelectronic transistors,functional protein arrays,and selfsupporting cell culture sheets.The dissertation on functional design,assembly and patterning of Csg A proteins for diverse applications will not only significantly enhance technological innovations in nanotechnologies,biomaterials,bioengineering,and biomanufacturing,but also stimulate new research efforts in harnessing diverse functional amyloids to develop new biomaterials,nanotechnologies,and devices.