FIBROUS PROTEINACEOUS NETWORKS AND METHODS OF USE THEREOF

REUATED APPUIC A TIONS

The instant application claims priority to U.S. Provisional Application No.

62/687,991, filed on June 21, 2018, the entire contents of which are expressly incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. 1410751 awarded by National Science Foundation. The government has certain rights in the invention.

BACKGROUND

Fibrous proteins such as collagen, elastin, keratin and fibrin perform key functions in the human body and are of high interest in the field of biomedical applications, especially in tissue engineering. Unfortunately, extraction, purification and synthesis of fibrous proteins can be difficult, time consuming, and expensive. For example, keratin extraction out of keratin-rich sources, like wool or hair, uses reducing agents containing thiols or oxidation methods which are and harmful to the environment and require a large amount of means, making those methods expensive. Moreover, chemicals used during keratin extraction are toxic and can damage the integrity of the extracted keratin. See Amin Shavandi et al., In: Biomaterials Science, 2017; David H Baker, The Journal of Nutrition 136.6, 2006; Mendal Friedman, Journal of Agricultural and Food Chemistry, 47.4, 1999.

Accordingly, there is a need for more efficient methods to extract or synthesize fibrous proteins, such as keratin and fibrin. Moreover, there is also a need for methods of production of bio-fabricated and environmentally friendly materials.

SUMMARY

Provided herein is a system which overcomes the known problems with

manufacturing of fibrous proteins and biofabricated proteinaceous networks for use in a variety of fields. Specifically, the present disclosure provides engineered or genetically altered microorganisms, such as E. coli, that produce fibrous proteinaceous networks, such as curli fibers, that are fused to fibrous proteins such as fibrin, elastin, keratin or collagen. The engineered fusion protein nanofibers disclosed herein enable the fibrous protein to be displayed on a bio film, without the need for purification or chemical modification. The fibrous proteins are co-expressed with amyloid nanofibers on the biofilm, and there is no need for chemical conjugation reagents. Moreover, the fusion proteins on the biofilm form a proteinaceous network that retains the mechanical properties and biological functions of the fibrous protein, therefore form a unique structure on the biofilm. The constructs disclosed herein are useful for creating a new material capable of, for example, being used as bacto- inks for three-dimensional printing (3D printing) and scaffolds for cell culturing in tissue engineering. The bacto-inks of the invention can further contain additional functional curli fibers or additional engineered bacteria that produce functional curli fibers. The bacto-inks containing functional curli fibers may be self-regenerating and have specific bioactivities. Such bacto-inks have applications in various fields such as biosensing, binding, signaling, and drug delivery.

In one aspect, the disclosure provides an amyloid fusion protein comprising an amyloid protein fused to one or more protein, or fragments thereof. In one embodiment, the one or protein is a fibrous protein. In one embodiment, the amyloid fusion protein is selected from the group consisting of CsgA and fragments thereof, A-beta, alpha- synuclein, TasA, and Sup35. In one embodiment, the amyloid fusion protein is CsgA, or a fragment thereof.

In one aspect, the disclosure provides an amyloid fusion protein comprising an amyloid protein fused to a fibrous protein. In one embodiment, the amyloid fusion protein is selected from the group consisting of CsgA and fragments thereof, A-beta, alpha- synuclein, TasA, and Sup35. In one embodiment, the amyloid fusion protein is CsgA, or a fragment thereof.

In one embodiment, the fibrous protein fused to the amyloid fusion protein is selected from the group consisting of keratin, elastin, fibrin and collagen, or a fragment thereof. In one embodiment, the fibrous protein fused to the amyloid fusion protein is fibrin, or a fragment thereof. In one embodiment, the fibrous protein fused to the amyloid fusion protein comprises an a chain of a fibrinogen. In one embodiment, the fibrous protein fused to the amyloid fusion protein comprises a sequence having at least 80% homology to SEQ ID NO:9. In one embodiment, the fibrous protein fused to the amyloid fusion protein comprises a g chain of a fibrinogen. In one embodiment, the fibrous protein fused to the amyloid fusion protein comprises a sequence having at least 80% homology to SEQ ID NO: 10.

In one embodiment, the fibrous protein fused to the amyloid fusion protein is a keratin. In one embodiment, the fibrous protein fused to the amyloid fusion protein is a K5 keratin. In one embodiment, the fibrous protein fused to the amyloid fusion protein comprises a sequence having at least 80% homology to SEQ ID NO: 11. In one embodiment, the fibrous protein fused to the amyloid fusion protein comprises a K14 keratin. In one embodiment, the fibrous protein fused to the amyloid fusion protein comprises a sequence having at least 80% homology to SEQ ID NO: 12.

In another aspect, the disclosure provides a plurality of amyloid fusion proteins comprising a first amyloid fusion protein and a second amyloid fusion protein, wherein the first amyloid fusion protein comprises a first amyloid protein fused to a first fibrous protein; and wherein the second amyloid fusion protein comprises a second amyloid protein fused to a second fibrous protein, wherein the first fibrous protein is capable of binding or is bound to the second fibrous protein.

In one embodiment, the first fibrous protein of the plurality of amyloid fusion proteins comprises an a chain of a fibrinogen, and the second fibrous protein comprises a g chain of a fibrinogen. In one embodiment, the first fibrous protein of the plurality of amyloid fusion proteins comprises a sequence having at least 80% homology to SEQ ID NO:9, and the second fibrous protein comprises a sequence having at least 80% homology to SEQ ID NO: 10.

In one embodiment, the first fibrous protein of the plurality of amyloid fusion proteins comprises a K5 keratin, and the second fibrous protein comprises a K14 keratin. In one embodiment, the first fibrous protein of the plurality of amyloid fusion proteins comprises a sequence having at least 80% homology to SEQ ID NO: 11, and the second fibrous protein comprises a sequence having at least 80% homology to SEQ ID NO: 12.

In one embodiment, the amyloid protein is selected from the group consisting of CsgA and fragments thereof, A-beta, alpha- synuclein, TasA, and Sup35. In one embodiment, the amyloid protein is CsgA, or a fragment thereof. In one embodiment, the CsgA, or fragment thereof, is an E. coli CsgA, or fragment thereof. In one embodiment, the E. coli CsgA comprises a sequence having at least 80% identity to SEQ ID NO:l.

In another aspect, the disclosure provides an isolated nucleic acid encoding any of the amyloid fusion proteins or the plurality of amyloid fusion proteins described herein.

In another aspect, the disclosure provides a vector comprising any of the isolated nucleic acids described herein.

In another aspect, the disclosure provides a curb fiber comprising any of the amyloid fusion proteins or the plurality of amyloid fusion proteins described herein.

In another aspect, the disclosure provides a fibrous proteinaceous network comprising any of the amyloid fusion proteins or the plurality of amyloid fusion proteins described herein. In another aspect, the disclosure provides an engineered microbial cell comprising any of the amyloid fusion proteins or the plurality of amyloid fusion proteins described herein.

In another aspect, the disclosure provides an engineered microbial cell comprising the any of the isolated nucleic acids described herein.

In another aspect, the disclosure provides an engineered microbial cell expressing any of the curli fibers described herein. In one embodiment, the engineered microbial cell is an E. coli cell.

In another aspect, the disclosure provides a biomaterial comprising any of the amyloid fusion proteins or the plurality of amyloid fusion proteins, the curli fiber, or the fibrous proteinaceous network described herein.

In one embodiment, the biomaterial further comprises an engineered microbial cell.

In one embodiment, the biomaterial further comprises any of the engineered microbial cells described herein.

In another aspect, the disclosure provides a hydrogel comprising any of the amyloid fusion proteins or the plurality of amyloid fusion proteins disclosed herein, the curli fiber disclosed herein, or the fibrous proteinaceous network disclosed herein.

In another aspect, the disclosure provides a bioink comprising any one of the biomaterials disclosed herein.

In another aspect, the disclosure provides a bioink comprising a hydrogel disclosed herein.

In another aspect, the disclosure provides a method of producing a biomaterial capable of forming a fibrous proteinaceous network, the method comprising culturing a first genetically engineered bacterium in culture media, wherein the first genetically engineered bacterium expresses a first amyloid fusion protein comprising a first amyloid protein and a first fibrous protein, culturing a second genetically engineered bacterium in the culture media, wherein the second genetically engineered bacterium expresses a second amyloid fusion protein comprising a second amyloid protein and a second fibrous protein, wherein the first fibrous protein binds to the second fibrous protein, thereby forming a plurality of curli fibers which form a biomaterial, thereby producing a biomaterial capable of forming a fibrous proteinaceous network.

In another aspect, the disclosure provides a method of producing a biomaterial capable of forming a fibrous proteinaceous network, the method comprising culturing a first genetically engineered bacterium in a first culture media, wherein the first genetically engineered bacterium expresses a first amyloid fusion protein comprising a first amyloid protein and a first fibrous protein, culturing a second genetically engineered bacterium in a second culture media, wherein the second genetically engineered bacterium expresses a second amyloid fusion protein comprising a second amyloid protein and a second fibrous protein, mixing the first culture media and the second culture media, wherein the first fibrous protein binds to the second fibrous protein, thereby forming a plurality of curli fibers which form a biomaterial, thereby producing a biomaterial capable of forming a fibrous

proteinaceous network.

In one embodiment, the first fibrous protein and the second fibrous protein of the method are selected from the group consisting of keratin, elastin, fibrin and collagen.

In one embodiment, the first fibrous protein and the second fibrous protein of the method are a fibrin. In one embodiment, the first fibrous protein of the method is an a chain of a fibrinogen, and the second fibrous protein of the method is a g chain of a fibrinogen. In one embodiment, the first fibrous protein of the method comprises a sequence having at least 80% homology to SEQ ID NO:9, and the second fibrous protein of the method comprises a sequence having at least 80% homology to SEQ ID NO: 10.

In one embodiment, the first fibrous protein and the second fibrous protein of the method are a keratin. In one embodiment, the first fibrous protein of the method is a K5 keratin, and the second fibrous protein of the method is a K14 keratin. In one embodiment, the first fibrous protein of the method comprises a sequence having at least 80% homology to SEQ ID NO: 11, and the second fibrous protein of the method comprises a sequence having at least 80% homology to SEQ ID NO: 12.

In one embodiment, the first amyloid protein and the second amyloid protein of the method are selected from the group consisting of CsgA and fragments thereof, A-beta, alpha- synuclein, TasA, and Sup35. In one embodiment, the first amyloid protein and the second amyloid protein of the method is CsgA, or a fragment thereof. In one embodiment, the CsgA, or fragment thereof of the method, is an E. coli CsgA, or fragment thereof. In one

embodiment, the E. coli CsgA of the method comprises a sequence having at least 80% identity to SEQ ID NO:l.

In one embodiment, the first genetically engineered bacterium and the second genetically engineered bacterium of the method are a first genetically engineered E. coli bacterium and a second genetically engineered E. coli bacterium.

In one embodiment, the method further comprises a step of removing the first genetically engineered bacterium and the second genetically engineered bacterium from the bio material. In one embodiment, the removing step of the method further comprises washing the first genetically engineered bacterium and the second genetically engineered bacterium from the biomaterial. In one embodiment, the removing step of the method comprises killing the first genetically engineered bacterium and the second genetically engineered bacterium in the bio material.

In one embodiment, the method further comprises forming a hydrogel.

In one embodiment, the method further comprises forming a bioink.

In another aspect, the disclosure provides use of any of the hydrogels or bioinks described herein as an ink for three-dimensional printing (“3D” printing).

In another aspect, the disclosure provides use of any of the hydrogels or bioinks disclosed herein as a sealant and scaffold for wound dressing or tissue engineering.

In another aspect, the disclosure provides a bioink comprising biomaterials selected from the group consisting of calcium hydroxyapatite, cellulose, chitin and silica.

Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIGs. 1A and IB depict concepts of network formation through mixing of two bacteria cultures (FIG. 1A) or co-culturing the matching bacteria (FIG. IB).

FIGs. 2A, 2B, 2C, 2D, 2E, and 2F depict fibrin fiber aggregation of mixed and co cultured method. All bacteria were expressing fibers for 48 hours in a shaking incubator for 48 hours, at 37 °C and 225 rpm. Bacterial ECM displaying CsgA-a or CsgA-g alone shows little aggregation of only CsgA-a fibers (FIG. 2A) or little aggregation of only CsgA-g fibers (FIG. 2B). Bacterial ECM displaying CsgA-ay _m ixed shows dense fiber aggregation of CsgA- aymixed fibers (FIGs. 2C-2E). Bacterial ECM displaying CsgA-ay _co -cuitured shows higher fiber aggregation of CsgA-ay _co -cuitured fibers after 48 hours (FIG. 2F).

FIGs. 3A 3B, 3C, 3D, 3E, and 3F depict keratin fiber aggregation of mixed and co culture method. All bacteria were expressing fibers for 48 hours in a shaking incubator for 48 hours, at 37°C and 225 rpm. Bacterial ECM displaying CsgA-K5 or CsgA-Kl4 alone shows little aggregation of only CsgA-K5 fibers (FIG. 3A) or little aggregation of only CsgA-Kl4 fibers (FIG. 3B). Bacterial ECM displaying CsgA-KSKH _mixed shows dense fiber aggregation of CsgA-KSKH _mixed fibers (FIGs. 3C-3E). Bacterial ECM displaying CsgA- K5 K 14 _co-cuitured shows higher fiber aggregation of CsgA-K5Kl4 _co-Cuitured fibers after 48 hours

(FIG. 3F).

FIGs. 4A, 4B, 4C, and 4D depict the micro structure of fibrin inspired curli hydrogels. FIG. 4A depicts CsgA-a based hydrogel showing a small pore size structure. FIG. 4B depicts CsgA-g based hydrogel with large pore size. FIG. 4C depicts formation of aligned looking fibers of CsgA-ay _miXC d based hydrogels. FIG. 4D depicts CsgA-ayco-cuitured based hydrogel, showing similar alignment as for CsgA-ay _{mi XC} d based hydrogels.

FIG. 5 depicts optical images of fibrin inspired curli hydrogels. From left to right are: CsgA-a based hydrogel, CsgA-g based hydrogel, CsgA-ay _{mi XC} d based hydrogels, and CsgA-ayco-cuitured based hydrogel, respectively.

FIG. 6 depicts the mechanical properties of the four different types of fibrin inspired hydrogels.

FIG. 7 depicts 3D structure construction with fibrin inspired hydrogels.

FIG. 8 depicts rheology properties of the 3D printing bacto-ink made from curli variants (Csg A-ay _miXCd or CsgA-ay _to-cuiiured ) without presence of bacteria or with bacteria (alpha-gamma live) or its single components alpha or gamma. The steady- state flow behavior of the inks is measured by viscosity curves at increasing and decreasing shear rates.

FIGs. 9A, 9B, 9C, and 9D depict 3D printing performance of CsgA-a (FIG. 9A), CsgA-g (FIG. 9B), CsgA-ay _miXed (FIG. 9C), and CsgA-ay _to.tU|lurcd (FIG. 9D), respectively.

3D printing performance was measured by the cross length of the printed filaments as a function of flow rate under a broad range of extrusion pressure.

FIGs. 10A, 10B, 10C, and 10D depict 3D printing using bacto-ink made from curli variant CsgA-ay _co -cuitured into a lO-layered circle (FIGs. 10A and 10B) or a lO-layered square (FIGs. 10C and 10D).

FIGs. 11A, 11B, and 11C are schematic illustrations of 3D printing inks made from fibrin inspired CsgA-a, CsgA-g, CsgA-ay _miXCd and/or CsgA-ay _to-cuiiured bacto-inks combined with functional curli fibers (FIG. 11A); combined with engineered living microbes (FIG. 11B); or combined with engineered living bacteria that can express functional curli fibers after printing and upon specific induction for secretion (FIG. 11C).

FIGs. 12A and 12B depict self-regeneration of 3D printing ink comprising of CsgA- ay _cocuitured and bacteria that produces CsgA-a and CsgA-g. Optical image of the grid after 1 hour (FIG. 12A) and 2 days (FIG. 12B) of printing. FIG. 13 depicts a capsule-like 3D printed pattern from a bacto-ink comprising CsgA- ay _cocuitured and bacteria that secrete an anti-cancer drug, azurin.

FIG. 14 depicts a spider-web like 3D printed pattern from a bacto-ink comprising CsgA-ay _cocuitured and bacteria that secrete a functional curli fiber comprising a BPA

(Bisphenol A) binding domain fused to the c-terminus of CsgA.

DETAILED DESCRIPTION

Disclosed herein are engineered bacteria which are capable of producing one or more nanofibers comprising a fusion protein. The engineered bacteria disclosed herein allow for the co-expression of amyloid nano fibers and a fibrous protein on the surface of a bio film, and the formation of fibrous proteinaceous networks on the biofilm. Such amyloid nanofibers and biofilms are useful for creating a new material capable of, for example, being used as bacto-inks for three-dimensional printing (3D printing) and scaffolds for cell culturing in tissue engineering. Aspects of the present disclosure use principles of Biofilm Integrated Nanofiber Display (BIND).

In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

Definitions

As used herein, an“amyloid”,“amyloidogenic protein”, or“amyloid-based structure” refers to an polymeric aggregate of amyloid polypeptides. In some embodiments, the amyloid-based structure forms a structure of fibrillary morphology. In some embodiments, the amyloid-based structure is a curli fiber. In some embodiments, the amyloid-based structure is formed by a heterogeneous population of amyloid polypeptides. In some embodiments, the amyloid-based structure is formed by a homogenous population of amyloid polypeptides. An amyloid-based structure may be formed by any population of amyloid polypeptides, including but not limited to CsgA, A-beta, alpha- synuclein, TasA, Sup35, or other functional amyloids derived from bacteria and fragments and mutants of CsgA. In one embodiment, the amyloid protein is CsgA. As used herein, the term“curli fiber” refers to the primary proteinaceous structural component of E. coli biofilms. Curli fibers are highly robust functional amyloid nanofibers with a diameter of ~4-7 nm that exist as extended tangled networks encapsulating the cells. Curli fibers are formed from the extracellular self-assembly of CsgA, a small secreted l3-kDa protein. A“plurality of curli fibers” refers to more than one curli fiber.

As used herein,“CsgA” refers to the major structural subunit of the curli fiber. The sequences of CsgA and its homologs are known in a number of species. For example, the sequence of E. coli CsgA is known (NCBI Gene ID NO: 949055; (polypeptide)). CsgA polypeptide (NCBI Ref Seq: NP_4l5560):

mkllkvaaiaaivfsgsalagvvpqyggggnhggggnnsgpnselniyqygggnsal alqtdarnsdltitqhgggngadvgqg sddssidltqrgfgnsatldqwngknsemtvkqfgggngaavdqtasnssvnvtqvgfgn natahqy (SEQ ID NO:l). A CsgA protein may include naturally occurring mutations or variants of CsgA, homologs of CsgA, or engineered mutations or variants of CsgA. In some embodiments,“CsgA” refers to E. coli CsgA. In some embodiments,“CsgA” refers to a polypeptide having at least 80% homology to SEQ ID NO:l ( e.g ., 80% or greater homology, 90% or greater homology, or 95% or greater homology).

As used herein,“fibrous protein” is a member of a class of long, filamentous proteins that form connective tissue, tendons, bone and muscle fiber in animal and human bodies. Fibrous proteins form“rod” or“wire”-like shapes and are usually inert structural or storage proteins. In one aspect, a fibrous protein is a full-length protein that contains multiple subunits that form a filament. In one aspect, a fibrous protein is a subunit or a polypeptide chain of a protein that forms a filament. In one aspect, a fibrous protein is a fragment of a subunit or a polypeptide chain of a protein that forms a filament. In some embodiments, a fibrous protein is a full-length keratin, a subunit of a keratin, or a fragment of a subunit of a keratin. In some embodiments, a fibrous protein is a full-length elastin, a subunit of an elastin or a fragment of a subunit of an elastin. In some embodiments, a fibrous protein is a full-length fibrin, a subunit of a fibrin or a fragment of a subunit of a fibrin. In other embodiments, a fibrous protein is a full-length collagen, a subunit of a collagen or a fragment of a subunit of a collagen.

The terms“protein" and“polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha- amino and carboxy groups of adjacent residues. The terms“protein,” and“polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms“protein” and“polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

A“nucleic acid” or“nucleic acid sequence” may be any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single- stranded or double-stranded. A single- stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single- stranded nucleic acid not derived from any double- stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

As used herein, the term“gene” refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5’ non coding sequences) and following (3’ non-coding sequences) the coding sequence. In one embodiment, a“gene” does not include regulatory sequences preceding and following the coding sequence. Each gene may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene may be present in the bacterium, wherein one or more copies of the gene may be altered as described herein.

A“native gene” refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence. A“chimeric gene” refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature.

As used herein, the term“endogenous gene” refers to a native gene in its natural location in the genome of an organism.

As used herein, a“heterologous” gene or“heterologous sequence” refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell. “Heterologous gene” includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature.

As used herein, the term“expression” refers to the transcription and stable

accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide.

As used herein, the term“plasmid” or“vector” refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell’s genome. Plasmids are usually circular and capable of autonomous replication. Plasmids may be low-copy, medium- copy, or high-copy, as is well known in the art. Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell. A plasmid may comprise a nucleic acid sequence encoding a heterologous gene or gene cassette.

As used herein, the term“transform” or“transformation” refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically- stable inheritance.

Host bacterial cells comprising the transformed nucleic acid fragment are referred to as “recombinant” or“transgenic” or“transformed” organisms.

As used herein, the term“engineered microbial cell” or“engineered bacterial cell” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, an engineered bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Engineered bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome. As used herein, the term“fusion”,“protein fusion” or“fusion protein” refers to a chimeric protein created through the joining of two or more genes that originally encoded separate proteins. A protein fusion is created artifically using recombinant DNA technology. Disclosed herein are amyloidogenic proteins fused, or linked, to a polypeptide (e.g., a firbous protein). In one aspect, the amyloidogenic protein is fused to a fibrous proetin. In one embodiment, a CsgA may be fused directly to a fibrous protein. In one embodiment, a CsgA may be fused to a subunit of a fibrous protein (e.g., an a chain of a fibrin, a g chain of a fibrin). In one embodiment, a CsgA may be fused to a fragment of a subunit of a firbous protein (e.g., a fragment of an a chain of a fibrin, a g chain of a fibrin, a fragment of K5 keratin or a fragment of K14 keratin). In another embodiment, a CsgA may be fused indirectly, e.g., by a linker, to a fibrous protein, a subunit of a fibrous protein or a fragment of a subunit of a firbous protein.

As used herein, the term“bound to” refers to an interaction between to molecules or proteins. A protein may be covalently or non-covalently bound to another protein or molecule. As used herein, a“covalent bond” refers to a chemical bond that involves the sharing of electron pairs between atoms. In contrast, a“non-covalent bond” does not involve the sharing of electrons, but involves more dispersed variations of electrmagnetic interactions between molecules. Non-covalent bonds include, but are not limited to, electrostatic, van der Walls forces, and hydrophobic effects.

As used herein the term“plurality” refers to more than one kind or type of a particular unit. A“plurality of curli fibers” refers to more than one type of curli fiber, wherein the curli fibers have different properties, functions and/or activities. For example, a plurality of curli fibers can be two types of curli fibers, wherein the first curli fiber has a first functionalizing polypeptide, and the second curli fiber has a second functionalizing polypeptide, wherein the first and second functionalizing polypeptides are different and confer different properties, functions and/or activities to the curli fibers.

As used herein, a“proteinaceous network” refers to a network formed by protein- protein interactions. In one aspect, a protenaceoud network is formed by the interactions betweeon fibrous proteins. In one aspect, a protenaceous network is formed by the interactions between amyloidogenic proteins. In one aspect, a protenaceous network is formed by the interactions between fibrous proteins and amyloidogenic proteins In one embodiment, a proteinaceous network is a fibrous network formed by the interactions between keratins. In one embodiment, a protenaceous network is a fibrous network formed by the interactions between fibrins. In one embodiment, a protenaceous network if a fibrous network formed by the interactions between fragments of keratins. In one embodiment, a protenaceous network is a fibrous network formed by the interactions between fragments of fibrins.

As used herein, a“hydrogel” is a macromolecular polymer gel constructed of a network of crosslinked polymer chains. In one embodiment, a hydrogel is a network of curli fibers fused to fibrous proteins. In one embodiment, a hydrogel is a biofilm comprising a proteinaceous network of curli fibers fused to fibrous proteins.

As used herein, a“linker” or a“spacer” refers to a polypeptide domain inserted between two proteins in a protein fusion. For example, a linker or a spcaer can be about 100 amino acids or less in size, about 75 amino acids or less in size, about 50 amino acids or less in size, about 40 amino acids or less in size, or smaller. In one aspect, linkers and spacers are inserted between a CsgA protein and a fibrous protein.

As used herein, the term“bio film matrix” refers to a matrix of extracellular polymeric substances, including, but not limited to extracellular DNA, proteins, glycopeptides, and polysaccharides, which was produced by a mass of microorganisms, such as bacteria, but wherein the microorganisms have been completely or almost completely killed or removed. Accordingly, in one embodiment, a“bio film matrix” does not comprise any microorganisms, such as bacteria. In one embodiment, a“bio film matrix” does not comprise any live microorganisms, such as bacteria.

As used herein, the term“bio film” refers to a matrix of extracellular polymeric substances, including, but not limited to extracellular DNA, proteins, glycopeptides, and polysaccharides, which are produced by a mass of microorganisms, such as bacteria. In one embodiment, a bio film comprises a bio film matrix and bacteria. In one embodiment, the bacteria are live bacteria.

As used herein, the term“biomaterial” refers to a natural or synthetic material made of multiple components which interacts with biological systems. In some embodiment, a biomaterial is a bio film. In some embodiments, a biomaterial is a bio film matrix. In some embodiments, a biomaterial is a hydrogel that comprises a fibrous proteinaceous network. In some embodiments, a biomaterial is a bioink used for 3D-printing.

As used herein, the term“bioinks” or“bacto-inks” refers to 3D printable inks fabricated or produced directly from genetically engineered bacteria. In some embodiments, a bioink is developed from a hydrogel comprising a fibrous proteinaceous network. In some embodiments, a bioink is developed from a hydrogel comprising functional and structural biomaterials such as calcium hydroxyapatite, cellulose, chitin and silica. In some embodiments, a bioink is BactoPrink.

As used herein,“BactoPrink” is a bioink employed to 3D print functional and structural biomaterials. In some embodiments, BactoPrink is a hydrogel comprising a fibrous proteinaceous network. In some embodiments, BactoPrink is developed from a hydrogel comprising functional and structural biomaterials such as calcium hydroxyapatite, cellulose, chitin and silica.

Definitions of common terms in cell biology and molecular biology can be found in The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN- 10: 0763766321); Kendrew et al. (eds.), , Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al, eds.

The articles“a” and“an,” as used herein, should be understood to mean“at least one,” unless clearly indicated to the contrary.

The phrase“and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example,“A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase“and/or” may be used interchangeably with“at least one of’ or“one or more of’ the elements in a list.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Curli Fibers

Curli fibers are the primary proteinaceous structural component of bio films. They are highly robust functional amyloid nano fibers with a diameter of ~4-7 nm that exist as extended tangled networks encapsulating the cells. Curli fibers are formed from the extracellular self-assembly of CsgA, a small secreted l3-kDa protein, see Chapman, M. R. el al. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295, 851-855 (2002), hereby incorporated by reference in its entirety. A homologous outer- membrane protein, CsgB, nucleates CsgA assembly and also anchors the nano fibers to the bacterial surface. Detached curli fibers can also exist as non-cell associated structural components of the extra-cellular membrane (ECM). The curli genes exist as two divergently transcribed operons ( csgBAC and csg DEFG), whose seven products mediate the structure (CsgA), nucleation (CsgB), processing (CsgE, F), secretion (CsgC, G), and direct transcriptional regulation (CsgD) of curli nanofibers. This curli secretion system is considered a distinct secretion system of its own in gram-negative bacterium and is named the Type- VIII secretion system (T8SS). See Desvaux et al, Trends Microbiol. 17, 139-45 (2009) hereby incorporated by reference in its entirety.

In one aspect, E. coli expressing curli fibers may be used for the methods disclosed herein. In another aspect, other useful bacteria with suitable secretions systems known to those of skill in the art may be used to produce the electrically conductive curli fibers of the present disclosure. The bacterium can be non-pathogenic.

As used herein,“CsgA” refers to the major structural subunit of the curli fiber. The sequences of CsgA and its homologs are known in a number of species, e.g., the sequence of E. coli CsgA is known (NCBI Gene ID NO: 949055; (polypeptide)). CsgA polypeptide (NCBI Ref Seq: NP_4l5560):

mkllkvaaiaaivfsgsalagvvpqyggggnhggggnnsgpnselniyqygggnsal alqtdarnsdltitqhgggngadvgqg sddssidltqrgfgnsatldqwngknsemtvkqfgggngaavdqtasnssvnvtqvgfgn natahqy (SEQ ID NO:l). In some embodiments,“CsgA” refers to E. coli CsgA. In some embodiments,“CsgA” refers to a polypeptide having at least 80% homology to SEQ ID NO:l (e.g., 80% or greater homology, 90% or greater homology, or 95% or greater homology), e.g., naturally occurring mutations or variants of CsgA, homo logs of CsgA, or engineered mutations or variants of CsgA.

As used herein, a“CsgA fusion” or an“engineered CsgA polypeptide” refers to a CsgA polypeptide comprising a heterologous polypeptide fused to the CsgA at either the C- terminus or the N terminus or both, but without interrupting the sequence of the CsgA polypeptide. In one aspect, a fibrous protein is fused to a CsgA protein at the C-terminus of the CsgA protein and it self-assembles into a curli fiber. A plurality of curli fibers diplaying one or more fibrous proteins or protein fragments is capable of forming a fibrous

proteinaceous network.

A“plurality of curli fibers” refers to more than one curli fiber. In one embodiment, a “plurality of curli fibers” refers to more than one type of curli fiber, wherein the curli fibers have different properties, functions and/or activities. For example, a plurality of curli fibers comprises two types of curli fibers, wherein the first curli fiber is fused to a first fibrous protein or a fragment of a fibrous protein, and the second curli fiber is bound to a second fibrous protein or a fragment of a fibrous protein, wherein the first and second fibrous proteins are different and confer different properties, functions and/or activities to each curli fiber. In some embodiments, a plurality of curli fibers comprises 2 different types of curli fibers.

Biofilms

Bacterial biofilms are constructed from protective nanoscale scaffolds of proteins, sugars, lipids, and extracellular DNA, and biofilms are self-generated protective structures that allow bacteria to adhere to both natural and man-made surfaces. See U.S. Patent No. 9,815,871, granted on November 14, 2017, the entire contents of which are expressly incorporated herein by reference in their entirety.

As used herein, the term“bio film” refers to a matrix of extracellular polymeric substances, including, but not limited to extracellular DNA, proteins, glycopeptides, and polysaccharides, which are produced by a mass of microorganisms, such as bacteria. The nature of a biofilm, such as its structure and composition, can depend on the particular species of bacteria present in the bio film. Bacteria present in a bio film are commonly genetically or phenotypically different than corresponding bacteria not in a bio film, such as isolated bacteria or bacteria in a colony. The biofilms disclosed herein are generally produced by culturing an engineered microbial cell comprising a CsgA fusion (and/or comprising a vector or nucleic acid encoding such a polypeptide) under conditions suitable for the production of curli fibers.

Conditions suitable for the production of a biofilm can include, but are not limited to, conditions under which a microbial cell is capable of logarithmic growth and/or polypeptide synthesis. Conditions may vary depending upon the species and strain of microbial cell selected. Conditions for the culture of microbial cells are well known in the art. Biofilm production can also be induced and/or enhanced by methods well known in the art, e.g., contacting cells with sub-inhibitory concentrations of beta- lactam or aminoglycoside antibiotics, exposing cells to fluid flow, contacting cells with exogenous poly-N- acetylglucosamine (PNAG), or contacting cells with quorum sensing signal molecules. In some embodiments, conditions suitable for the production of a bio film can also include conditions which increase the expression and secretion of CsgA, e.g., by exogenously expressing CsgD. In one embodiment, a bio film refers to the matrix of extracellular polymeric substances that is produced by a mass of microorganisms, wherein the bacteria have been killed or removed. In one embodiment, a bio film does not comprise any bacteria. In another embodiment, a bio film does not comprise any live bacteria. In some embodiments, the bio film can further include the bacterium which produced the bio film.

Biofilms disclosed herein may be produced by genetically engineering or modifying bacteria to comprise a nucleic acid encoding a CsgA fusion, consisting of a CsgA protein linked to a fibrous protein ( e.g ., keratin, elastin, fibrin or collagen), and growing the engineered bacteria in situ or in culture media. The nucleic acid encoding a CsgA protein fused to the fibrous protein may be heterologous and introduced into the bacterium using methods known to those of skill in the art. The nucleic acid encoding a fusion CsgA protein may result from mutation of the endogenous nucleic acid encoding CsgA using methods known to those of skill in the art. In one embodiment, the biofilm is produced by engineered bacteria comprising a nucleic acid encoding CsgA protein fused to a fibrous protein. The CsgA protein may be fused to the fibrous with a linker domain in between.

A“vector” includes a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. A vector can be viral or non-viral. Many vectors useful for transferring genes into target cells are available, e.g., the vectors may be episomal, e.g., plasmids, virus derived vectors or may be integrated into the target cell genome, through homologous recombination or random integration. In some embodiments, a vector can be an expression vector. An“expression vector” can be a vector that has the ability to incorporate and express heterologous nucleic acid fragments in a cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms. The nucleic acid incorporated into the vector can be operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.

In some embodiments, a nucleic acid encoding a CsgA fusion can be present within a portion of a plasmid. Plasmid vectors can include, but are not limited to, pBR322, pBR325, PACYC177, pACYCl84, pUC8, pUC9, pUCl8, pUCl9, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see“Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH82l, pGEX, pET series (see Studier et. al,“Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology, vol. 185 (1990), which is hereby incorporated by reference in its entirety). In one embodiment, the plasmid vector is a pET2ld plasmid. In one embodiment, a nucleic acid encoding a CsgA fused to a fibrous protein is present in a pET2ld plasmid.

The engineered bacteria can secrete the CsgA fusion, which results in curli fiber production, followed by biofilm formation. Specifically, after secretion, the CsgA fusion is nucleated to form a self-assembling amyloid at the cell surface, and then continues to polymerize into long fibers that eventually encapsulate the cells and provide the biofilm with structural support. In one embodiment, the CsgA is fused to a fibrous protein, resulting in the formation of a fibrous proteinaceous network. In one embodiment, the fibrous protein is keratin. In one embodiment, the fibrous protein is elastin. In one embodiment, the fibrous protein is fibrin. In another embodiment, the fibrous protein is collagen.

A bacterial cell described herein can be of any species. Preferably, the bacterial cells are of a species and/or strain which is amenable to culture and genetic manipulation. In some embodiments, the bacterial cell can be a gram-positive bacterial cell. In some embodiments, the bacterial cell can be a gram-negative bacterial cell. In some embodiments, the parental strain of the bacterial cell of the technology described herein can be a strain optimized for protein expression. Non- limiting examples of bacterial species and strains suitable for use in the present technologies include Escherichia coli, E. coli BL21, E. coli Tuner, E. coli Rosetta, E. coli JM101, and derivatives of any of the foregoing. Bacterial strains for protein expression are commercially available, e.g., EXPRESS™ Competent E. coli (Cat. No.

C2523; New England Biosciences; Ipswich, MA). In one embodiment, the curli fibers are produced by engineered or non-naturally occurring bacterium. In one embodiment, the bacterium is E. coli. In one embodiment, the bacterium is PNQ4 ( E . coli strain derived from LSR10 (MC4100, ACsgA, k(DE3), CamR)) which was constructed to knockout the curli operon. In one embodiment, the bacterium is non-pathogenic.

In one embodiment, disclosed herein are methods of producing a bio film by culturing an engineered bacteria in culture media, wherein the engineered bacteria comprises a nucleic acid sequence encoding a curli fiber fused to a fibrous protein. In one embodiment, the fibrous protein is keratin. In one embodiment, the fibrous protein is elastin. In one embodiment, the fibrous protein is fibrin. In another embodiment, the fibrous protein is collagen. Fibrous Proteins and Formation of Fibrous Proteinaceous Networks

Fibrous proteins such as collagen, elastin, keratin and fibrin form networks and have various important functions in the human body. They contribute to the formation of connective tissues, are crucial for wound healing and blood coagulation, and provide structural support for cells in form of intermediate filaments (IFs). Furthermore, fibrous proteins are major components of the ECM and contribute to its structure and stiffness, see Christian Frantz, Kathleen M Stewart, and Valerie M Weaver,“The extracellular matrix at a glance,” J Cell Sci 123.24 (2010), pp. 4195-4200. Cell-matrix interaction is of great importance to many physiological processes ranging from cell communication, motility, migration, cell fate and morphology. See DL Humphries, JA Grogan, and EA Gaffney, “Mechanical Cell-Cell Communication in Fibrous Networks: The Importance of Network Geometry,” Bulletin of mathematical biology 79.3 (2017), pp. 498-524. Furthermore, an intact ECM is vital for wound healing and essential in the field of tissue engineering, where engineered biocompatible scaffolds are often formulated with naturally existing ECM proteins. See Youhwan Kim et al.“Extracellular Matrix Revisited: Roles in Tissue

Engineering,” International neurourology journal 20.Suppl 1 (2016), S23. Consequently, abnormal ECM structures can lead to congenital defects, deregulated cell proliferation or loss of cell differentiation, and is linked to altered intrinsic cell function. See Pengfei Lu et al. “Extracellular matrix degradation and remodeling in development and disease,” Cold Spring Harbor perspectives in biology 3.12 (2011), a005058; Shelly R Peyton et al.“The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function,” Cell biochemistry and biophysics 47.2 (2007), pp. 300-320. Thus, it takes a role in many pathological processes, including fibrosis and tumor invasion. See Pengfei Lu et al.

“Extracellular matrix degradation and remodeling in development and disease,” Cold Spring Harbor perspectives in biology 3.12 (2011), a005058.

Any of known fibrous protein can be utilized in the CsgA- fibrous protein fusions disclosed herein. In some embodiments, the fibrous protein is keratin. Keratin is used for the production of hair or nails, but is also playing an important role for the structural framework of the cell in form of intermediate filaments. See GM Cooper. The Cell: A Molecular Approach, 2nd edn. The Cell: A Molecular Approach, Sunderland, MA. 2000; Chang-Hun Lee et al.“Structural basis for heteromeric assembly and perinuclear organization of keratin filaments,” Nature structural & molecular biology 19.7 (2012), pp. 707-715. Keratin provides structural support to the cells and allows them to migrate, proliferate and differentiate, and keratin intermediate filaments are formed through copolymerization of one acidic (type I) and one basic (type II) keratin protein, which are synthesized by epithelial cells. See Chang-Hun Lee et al.“Structural basis for heteromeric assembly and perinuclear organization of keratin filaments,” Nature structural & molecular biology 19.7 (2012), pp. 707-715.

The first step in the development of intermediate filaments is the formation of dimers, characterized through a coiled coil structure of two polypeptide chains (here acidic and basic keratin) that are wound together. As the dimers associate in a staggered antiparallel manner, they form tetramers, which can further assemble to form proto filaments. Protofilaments finally wind around each other to form intermediate filaments. See GM Cooper. The Cell: A Molecular Approach, 2nd edn. The Cell: A Molecular Approach, Sunderland, MA. 2000; Sergei V Strelkov et al.“Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly,” The EMBO journal 21.6 (2002), pp. 1255-1266; Peter M Steinert et al.“Keratin intermediate filament structure: crosslinking studies yield quantitative information on molecular dimensions and mechanism of assembly,” Journal of molecular biology 230.2 (1993), pp. 436-452.

The mechanical resilience of intermediate filament networks is crucial for the structural support of the cell. See JM Bonifas, AL Rothman, and EH Epstein Jr.

“Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities,” Science 254.5035 (1991), p. 1202. The integrity of those filaments is depending mainly on two factors. On one side, from concentration and length of the filaments. On the other side, from stable linkages between the filaments. The most studied keratin pair is keratin "K5" (type II) and "K14" (type I). This pair forms extremely stable tetramer subunits and organizes into cross-linked intermediate filament networks through interaction of the distal half of Kl4’s tail domain and two distinct regions in K5’s rod domain. See Pierre A

Coulombe and Elaine Fuchs.“Elucidating the early stages of keratin filament assembly,” The Journal of Cell Biology 111.1 (1990), pp. 153-169. The interface of K5-K14 coiled-coil heterodimer is characterized through non-covalent linkages of electrostatic interaction, hydrophobic interactions and hydrogen bonds as shown in picture 1. Mutations in either K5 or K14 are directly involved in a broad range of diseases, including many epithelial blistering disorders like epidermolysis bullosa simplex, underlying the importance of keratin’s intracellular function. See JM Bonifas, AL Rothman, and EH Epstein Jr.“Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities,” Science 254.5035 (1991), p. 1202. Besides the central role in the structural support of the cell, keratin has gained a greater interest in the field of biomedical engineering over the past decades. Keratin has the ability to facilitate cell adhesion, cell proliferation and promote tissue regeneration by creating cell binding domains such like fibronectin. See Mira Park et al.“Effect of discarded keratin-based biocomposite hydrogels on the wound healing process in vivo,” In: Materials Science and Engineering: C 55 (2015), pp. 88-94; Paulina Sierpinski et al.“The use of keratin biomaterials derived from human hair for the promotion of rapid regeneration of peripheral nerves,” Biomaterials 29.1 (2008), pp. 118-128; Amin Shavandi et al.

“Dissolution, Extraction and Biomedical Application of Keratin: Methods and Factors Affecting the Extraction and Physicochemical Properties of Keratin,” Biomaterials Science (2017). Moreover, keratin can function as a synthetic ECM due to its biodegradability and biocompatibility. Thus, keratin-based biomaterials have the potential to be a suitable platform for tissue engineering applications.

In one embodiment, the CsgA is fused to a full-length keratin. In one embodiment, the CsgA is fused to a fragment or a motif of a subunit of a keratin. In one embodiment, the CsgA is fused to K5. In another embodiment, the CsgA is fused to K14.

In some embodiments, the fibrous protein is fibrin. Fibrin is responsible for blood coagulation and therefore a key protein in human body. Additionally, fibrin provides an excellent scaffold for fibroblast adhesion and proliferation, and formation of granulation tissue. Therefore, fibrin contributes significantly in the process of wound healing. See Richard AF Clark.“Fibrin and wound healing,” Annals of the New York Academy of Sciences 936.1 (2001), pp. 355-367. Furthermore, fibrin derived polymer, fibrin glue, has a great importance in surgery where it can be used as a tissue glue and act as an alternative to sutures. See Anita Panda et al.“Fibrin glue in ophthalmology,” Indian Journal of

Ophthalmology 57.5 (2009), p. 371. The formation of fibrin is the final step in the coagulation cascade. Once the coagulation cascade is triggered, activated factor X

hydrolyses prothrombin to thrombin. Thrombin, for its part, triggers the formation of fibrin monomers by cleaving fibrinopeptide A and B (FpA and FpB) from the fibrinogen backbone. Fibrinogen is made up of 6 paired polypeptide chains (Aa) ₂ , (B/¾, g ₂ and is 45nm long. It is comprised of two outer D regions (each containing b - and g- nodule) that are connected to the central E region by a coiled-coil segment. See MW Mosesson.“Fibrinogen and fibrin structure and functions,” Journal of Thrombosis and Haemostasis 3.8 (2005), pp. 1894-1904.

However, the release of FpA exposes an a-chain motif GPR (Glycine - Proline - Arginine) called knob“A”. Knob“A” binds to an exposed hole“a” in the globular g-nodules of another fibrin monomer, resulting in a non-covalent knob-hole interaction. See Michael S Kostelansky et al.“2.8 A crystal structures of recombinant fibrinogen fragment D with and without two peptide ligands: GHRP binding to the“b” site disrupts its nearby calcium binding site,” Biochemistry 41.40 (2002), pp. 12124-12132. The exposure of knob“A” is essential and also sufficient to form fibrin. See Artem Zhmurov et al.“Structural basis of interfacial flexibility in fibrin oligomers,” Structure 24.11 (2016), pp. 1907-1917. The cleavage of FpB exposes a //-chain motif GHR (Glycine - Histidine - Arginine) called knob “B” that binds to its corresponding hole“b” (also non-covalently) in the globular //-nodule of another fibrin monomer. FpA cleavage is faster than FpB. See Harold A Scheraga and Michael Laskowski.“The fibrinogen-fibrin conversion,” Advances in protein chemistry 12 (1957), pp. 1-131. Fibrin polymerization is initiated when two fibrin monomers form a half- staggered dimer. The monomers are hold together by the A:a knob-hole interaction of Knob “A” fitting into hole“a”. The addition of a third fibrin molecule to the half- staggered dimer forms an end-to-end connection of two fibrin’s lateral D regions. See Stephen J Everse et al. “Crystal structure of fragment double-D from human fibrin with two different bound ligands,” Biochemistry 37.24 (1998), pp. 8637-8642. This weak D:D interface compromises the monomer junction in each of two fibrin oligomers. See John W Weisel and Rustem I Litvinov,“Mechanisms of fibrin polymerization and clinical implications,” Blood 121.10 (2013), pp. 1712-1719. Other fibrin monomers can add longitudinally to the dimers to form longer oligomers. Two oligomer strands can then laterally interact. See WE Fowler et al. “Structure of the fibrin protofibril,” Proceedings of the National Academy of Sciences 78.8 (1981), pp. 4872-4876. This interaction is mediated by the central E region of one fibrin monomer and two lateral D regions of two other fibrin molecules. The D-E-D structure is mainly held together by A-a knob-hole bonds.

Elongation of fibrin oligomers, results in the formation of protofibrils, which can associate with each other and aggregate laterally to form fibers. See Olga Kononova et al. “Molecular mechanisms, thermodynamics, and dissociation kinetics of knob-hole interactions in fibrin,” Journal of Biological Chemistry 288.31 (2013), pp. 22681-22692. For the formation of a 3- dimensional fibrin network, it is necessary for the fibers to branch. See John W Weisel.“Fibrinogen and fibrin,” Advances in protein chemistry 70 (2005), pp. 247- 299. Furthermore, the fibrin network is covalently cross-linked by factor XHIa, a plasma transglutaminase that is activated by thrombin in the presence of Ca ²⁺ . This results in stabilization of the fibrin network. See Laszlo Lorand.“Factor XIII: structure, activation, and interactions with fibrinogen and fibrin,” Annals of the New York Academy of Sciences 936.1 (2001), pp. 291-311.

In one embodiment, the CsgA is fused to a full-length fibrin. In one embodiment, the CsgA is fused to a subunit or a polypeptide chain of a fibrin (e.g., the a chain, g chain or b chain of fibrin). In one embodiment, the CsgA is fused to a fragment or a motif of a subunit of a fibrin.

Sequences of exemplary fibrous proteins and fibrous protein subunits are listed in Table 1 below.

Table 1. Exemplary sequences of fibrous proteins, fragments, or motifs of fibrous proteins

In one embodiment, the fibrous protein may comprise any of the fibrous protein sequences disclosed herein. In one embodiment, the fibrous protein may consist of any of the fibrous protein sequences disclosed herein. In one embodiment, the fibrous protein may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the fibrous protein sequences disclosed herein.

Using genetically modified bacteria ( e.g E. coli ) comprising CsgA-fibrous protein fusions described herein, a fibrous proteinaceous network can be formed. For example, a proteinaceous network described herein may comprise more than one type of curli fiber, each comprising a CsgA protein fused to a different fibrous protein, wherein a first CsgA fusion comprises a first fibrous protein non-covalently bound to a second fibrous protein of a second CsgA fusion, to form fibrous structures and proteinaceous networks.

In one embodiment, the fibrous proteinaceous network described herein comprises a first curli fiber wherein the CsgA protein is fused to a K5 keratin and a second curli fiber wherein the CsgA protein is fused to a K14 keratin, wherein the K5 protein is non-covalently bound to the K14 protein to form a fibrous structure and a proteinaceous network. In one embodiment, the K5 protein comprises the polypeptide of SEQ ID NO: 11. In one

embodiment, the K14 protein comprises the polypeptide of SEQ ID NO: 12.

In one embodiment, the fibrous proteinaceous network described herein comprises a first curli fiber wherein the CsgA protein is fused to the a chain (or a fragment or motif) of a fibrin and a second curli fiber wherein the CsgA protein is fused to a g chain (or a fragment or motif) of a fibrin, wherein the a chain motif is non-covalently bound to the g chain motif to form a fibrous structure and a proteinaceous network. In one embodiment, the a chain motif of fibrin is Knob“A”. In one embodiment, the a chain motif of fibrin is the polypeptide of SEQ ID NO:9. In one embodiment, the g chain motif of fibrin is hole“a”. In one

embodiment, the g chain motif of fibrin is the polypeptide of SEQ ID NO: 10.

Hydrogels

In one aspect, provided herein is a method of making a biologic hydrogel comprising contacting a liquid composition comprising a bacterial cell that expresses a curli fiber with a solubilization agent, thereby creating a mixture; contacting the mixture with a filter;

contacting the mixture with a surfactant; incubating the mixture, thereby allowing the mixture to gelate; and concentrating the mixture using filtration; thereby making the biologic hydrogel. In some embodiments, the solubilization agent can be a denaturing solubilization agent, a non-denaturing solubilization agent, or a mild denaturing solubilization agent. In some embodiments, the solubilization agent can be, but is not limited to, guanidine, urea, DMSO, SDS, b-mercaptoethanol, or n-propanol. In some embodiments, the solubilization agent is any agent or reagent capable of inducing lysis of a microbial cell ( e.g ., a bacterial cell) including a lysis buffer, lysozyme, a base such as sodium hydroxide, and others. The concentration of the solubilization agent that is used may be varied, and without wishing to be bound by any particular theory, may affect the purity of the amyloid fibers that are ultimately obtained using the methods described herein. In some embodiments, the solubilization agent is used at a concentration capable of inducing lysis of a bacterial cell. One of ordinary skill may readily ascertain the concentration of the solubilization agent necessary in order to induce lysis of a bacterial cell. For example, when guanidine hydrochloride (GdmCl) is the solubilization agent that is used in the methods described herein, the concentration of guanidine hydrochloride may range from 0.1 - 10 M. In some embodiments, the concentration of guanidine hydrochloride is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 2 M, about 3 M, about 4.0 M, about 5.0 M, about 6.0 M, about 7.0 M, about 8.0 M, about 9.0 M, about 10.0 M, or more.

In some embodiments, the methods described herein may be performed using vacuum filtration (e.g., using vacuum generated with a pump). In some embodiments, the methods described herein may be performed using gravity filtration. In some embodiments, the methods described herein may be performed using centrifugal filtration. In some

embodiments, the methods described herein may be performed using filter plates for small scale purification. The filtration set-ups used in the methods described herein may include vacuum filtration holders, butchner funnels, tabletop filtration systems, and the like.

In some embodiments, the filter is a filter membrane, a mesh, or a porous cloth. In one embodiment, the filter membrane is polycarbonate, nylon, cellulose,

polytetrafluoroethylene (Teflon ), polyethersulfone, polyvinylidene fluoride, or

polyvinylidene chloride. In one embodiment, the filter membrane is a hydrophilic nylon net. In one embodiment, the mesh is a metal mesh, a glass mesh, a ceramic mesh, a plastic mesh, or a polymer mesh.

In some embodiments, the filter membranes used in the presently disclosed methods may be, but are not limited to, polymer membranes made of polycarbonate, nylon, cellulose,

Teflon , polyethersulfone, polyvinylidene fluoride, polyvinylidene chloride, or other materials. In some embodiments, curli fiber aggregates can be filtered on cloths, or any other fabrics with pores of the appropriate size, as described herein. In some embodiments, curli fiber aggregates can be filtered on porous mesh with pores of the appropriate size, as described herein, such as, but not limited to, metal meshes, plastic meshes, glass meshes, ceramic meshes, or polymer meshes.

The filter membranes can have a pores of any shape or geometry. For example, in some embodiments, the pores can be circular. In other embodiments, the pores can be mesh- like. In some embodiments, the filter membranes can have pores of more than one shape or geometry.

The filter membrane may be of any geometric ( e.g ., circular, octagonal, rectangular, squared) or non- structured shape. The filter membrane may be of any size. For example, larger filters allow for scaling-up of the purification process.

In some embodiments, the surfactant can be an ionic surfactant or a non-ionic surfactant. In some embodiments, the surfactant can be, but is not limited to, SDS, 4- octylphenol polyethoxylate (also known as Triton X-100 ), polyethylene glycol sorbitan monolaurate (also known as Tween 20), polyethylene glycol sorbitan monooleate (also known as Tween 80). For example, when the surfactant is SDS, a solution comprising 1% (w/v), 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more may be used. In some

embodiments, the surfactant is SDS at a concentration of 5% (w/v).

In some embodiments, the methods further comprising air-drying the biologic hydrogel. In some embodiments, the air-drying is performed at room temperature for at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, or more.

In some embodiments, the method further comprises dehydrating the biologic hydrogel. In some embodiments, the method further comprises rehydrating the biologic hydrogel.

In some embodiments, the method further comprises imprinting the surface of the biologic hydrogel with a mold. In some embodiments, the mold is a nano mold. In some embodiments, the mold is a micro mold. In some embodiments, the mold comprises a pattern (e.g., a mesh or a structured surface). In some embodiments, the mold is a naturally occurring material.

In some embodiments, the filter membranes may be, but are not limited to, polymer membranes made of polycarbonate, nylon, cellulose, Teflon, polyethersulfone,

polyvinylidene, fluoride, Polyvinylidene chloride, or other materials (e.g., chitin, chitosan, alginate). In some embodiments, the filter membrane can have pores of any size larger than the size of a bacterium and smaller than the size of an aggregate of curli fibers

(approximately ~2 to 50 pm).

The methods described herein may be performed at a small scale ( i.e ., as a batch process) or may be adapted for large scale production of hydrogels.

In some embodiments, the hydrogel comprises a fibrous proteinaceous network. In some embodiments, the hydrogel can be used as an ink for 3D printing.

Methods and Uses

Disclosed herein is a versatile platform to create fibrous proteinaceous network of the extracellular matrix of bacteria, e.g., E. coli. Fibrous proteins, e.g., keratin and fibrin, fused to the curli fibers are immobilized site-specifically onto a biofilm surface, for example, with no need of any protein purification. Additionally, the fibrous proteinaceous networks, are formed in a self-assembling manner, for example, where no additional chemical conjugation is needed. The engineered biofilm is capable of forming and retaining a fibrous

proteinaceous network.

The compositions and methods described herein are useful in various applications.

By resembling mechanic and rheological properties of fibrous proteins, the proposed system would be biocompatible and have a wide range of applications in the fields of biomedical engineering and tissue engineering. Because of the stiffness and viscosity of the hydrogel produced, it could be used for instance as inks for three-dimensional printing (3D printing). Furthermore, the hydrogels can behave as a viscoelastic glue that has the potential to function as a tissue sealant like fibrin glue.

3D-Printing Inks

The fibrous proteinaceous networks, biofilms, hydrogels or biomaterials of the current invention can be used as inks for three-dimensional printing (3D printing). In some embodiments, the 3D-printing inks, e.g., bacto-inks, or Bacto Prinks can further comprise a functional curli fiber or an engineered microbial cell, e.g., a bacterium that expresses one or more functional curli fibers. In some embodiments, a functional curli fiber is a fusion protein of CsgA linked to a non-native functional polypeptide. In some embodiments, the CsgA protein is linked to the non-native functional polypeptide by a linker.

In some embodiments, the bacto-inks of the invention comprises a fibrous

proteinaceous network, biofilm, hydrogel or biomaterial of the current invention and is further mixed and contacted with a functional curli fiber. In some embodiment, the functional curli fiber is an isolated curli fiber. In some embodiments, the bacto-inks of the invention comprises a fibrous proteinaceous network, biofilm, hydrogel or biomaterial of the current invention and is further mixed and contacted with an engineered microbial cell, e.g., a bacterium that express a functional curli fiber. In some embodiments, the engineered bacterium comprises a nucleotide encoding the functional curli fiber.

As used herein, a functional polypeptide includes a polypeptide having an activity or function, such that when it is present in a bio film, it confers upon the bio film a property, function, or activity which it did not have in the absence of the activity of the polypeptide. Accordingly, an activity polypeptide can be, e.g. an enzyme, a polypeptide that binds another molecule, a binding domain, a peptide that is bound by another molecule (e.g. a ligand or epitope), or the like. Examples of polypeptides for use as activity polypeptides include, but are not limited to bisphenol A (BPA) binding domain, Metal binding domain (MBD);

SpyTag; graphene binding (GBP); carbon nanotube binding (CBP); gold binding (A3); CT43; FLAG; Z8; E14; QBP1; CLP12; and AFP8. In some embodiment, the non-native functional polypeptide is a therapeutic polypeptide, a diagnostic polypeptide, a tissue-binding polypeptide, a cell-binding polypeptide, an antimicrobial polypeptide, an anticancer polypeptide, an anti-inflammatory polypeptide, a polymer binding polypeptide, a metabolite binding polypeptide, a targeting polypeptide or a polypeptide that is a first pair of a binding pair of molecules. In some embodiments, the non-native functional polypeptide is not a fibrous protein, or a fragment thereof, that is contained in the amyloid fusion protein, or the fibrous proteinaceous network disclosed in the previous sections of current disclosure. In some embodiments, the functional curli fibers or engineered bacteria that produce the functional curli fibers are added to the bacto-ink in addition to the fibrous proteinaceous network, biofilm, hydrogel or biomaterial described in the previous sections of the current disclosure.

According to certain aspects, the functional polypeptide when present as part of an engineered CsgA polypeptide, is functional. A polypeptide is said to be“functional" or expressed as a“functional" polypeptide if the polypeptide retains at least about 50% of the activity (e.g. enzymatic activity or binding activity) that it has as an isolated polypeptide.

One of skill in the art can readily detect increases in reaction products and/or detect decreases in reaction substrates, e.g. by mass spectroscopy (MS, including, e.g., MADLI/TOF,

SELDI/TOF, LC-MS, GC-MS, HPLC-MS, etc., among others) or detect increases or decrease in binding to a binding partner, e.g. by immunoassays. In some embodiments, a functional activity polypeptide can retain at least 50% of the activity of the isolated polypeptide, e.g. 50% or more of the activity, 60% or more of the activity, 75% or more of the activity, or 90% or more of the activity of the isolated polypeptide.

Exemplary functional polypeptides and methods of making an engineered bacterium that expresses a fusion protein of CsgA with a non-native functional polypeptide are disclosed in, e.g., U.S. Patent No. US 2018/0258435 Al, and U.S. Patent No. 9,815,871, the entire content of each is hereby incorporated by reference.

In some embodiments, the bacto-ink disclosed herein is capable of self-regeneration due to the existence of live bacteria that produce the fibrous proteinaceous networks, biofilms, hydrogels or biomaterials. In some embodiment, the bacto-ink disclosed herein is capable of self-regeneration due to the existence of live bacteria that produce the functional curli fibers comprising the fusion protein of CsgA and the non-native functional polypeptide.

In one aspect, the composition and methods described herein can be used as a novel material for 3D printing. In some embodiments, the composition and methods are used as biocompatible materials for tissue engineering, such as tissue sealant. In some embodiments, the composition and methods are used in medical procedures such as bone replacement.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure.

Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

EXAMPLES

The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims.

Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al, Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning

Techniques Vol.l52, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); and Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al, ed., John Wiley and Sons, Inc.), which are all incorporated by reference herein in their entireties.

Example 1: Background and Introduction

Curli fibers can constitute up to 40% of the biomass of many biofilms and can therefore be engineered to create a biofilm, expressing the added functionalization in a large portion of the resulting engineered biofilm. E. coli derived curli fibers are one of the most studied type of amyloids which is therefore an advantage in comparison with other, less known types of amyloids produced by many other bacteria.

BIND works by exploiting this system. A laboratory strain of E. coli containing a deletion of the CsgA gene ( E . coli D cgsA) is transformed with a plasmid encoding an engineered version of the CsgA protein, which can be functionalized in a variety of ways by fusing different peptide domains (Nguyen et al., Nature Communications , 5, 4945, 2014). In other words, the CsgA protein can be engineered in order to display different types of coating treatments. It represents an easier and more flexible method compared to engineering the exopolysaccharide part of the biofilm, something that would require a multiple step pathway and which would have less chemical tolerance.

By genetic engineered biofilm matrix proteins of E. coli bacteria, it was possible to mimic prominent fibrous proteinaceous networks in the human body. It was hypothesized that through integration of engineered ECM of two reprogrammed cell lines, proteins derived from fibrous proteins such as fibrin or keratin displayed on curli fibers will be able to interact and aggregate to form the novel network. As for fibrin, the natural occurring network is imitated by the interaction of fused“a” and“g”, and therefore by the knob-hole bond. The coiled-coil structure in natural fibrin is replaced by CsgA that acts as the backbone for“a” and“g”. The same principal applies for keratin. Here the direct interaction of fused "K5" and "K14" onto curli fibers will form the coiled-coil heterodimers.

Example 2: Materials and Methods

Cell Strains. Plasmids and Reagents

The genes encoding CsgA-a, CsgA-g, CsgA-K5 and CsgA-Kl4 were synthesized (Integrated DNA Technologies) and cloned by overlap extension into pET2ld vectors consisting of the csgACEFG operon under the control of the T7 promoter (see Table 2 for sequences). All cloning was performed using isothermal Gibson Assembly cloning kit®, and verified by DNA sequencing. (Dorval Courchesne et al., ACS Biomaterials Science & Engineering , acsbiomaterials.6b00437, 2016), with a single operon, csgBACEFG, under the control of the T7 promoter, where the nucleator protein CsgB, responsible for connecting curli fibers to the external surface of the bacteria, was deleted. This plasmid was created using the two divergent operons regions, the csgBAC and csgEFG, which were PCR isolated from the W3110 strain of E. coli K12 and cloned by overlap extension into the pET2ld plasmid.

Table 2: Plasmids

The fusion expression of a, g, K5 and K14 onto curli fibers was performed in a curli operon deletion mutant of an E. coli strain, PQN4. The PQN4 is an E. coli strain derived from LSR10 (MC4100, ACsgA, k(DE3), CamR) which was constructed to knockout the curli operon. Table 3 provides a list of bacterial strains used in the Examples.

Table 3: Bacterial Strains Bacteria Cultivation and Curli Fiber Expression

300 mΐ of cloned PQN4 cells carrying the engineered plasmids, were seeded onto lysogeny broth (LB) agar plates containing 2% glucose and 100 pg/ml of carbenicillin. The bacteria were incubated over night at 37°C. The settled plates were stored upside down in a refrigerator at 4°C for a maximum of two weeks. Inoculation of the reprogrammed bacteria cultures was performed using a 1:10:1000 mixture of LB, 20% (v/v) glucose and 98% (v/v) carbenicillin. 5ml of this mixture were filled into a tube.

For the expression of a single protein, a reprogrammed bacterial colony was loaded onto a tip of a pipette and put into a falcon tube along with the antibiotic and glucose containing mixture. The tubes were then incubated for four to six hours or overnight in a shaking incubator at 37°C and 225 revolutions per minute (rpm). Next, the content of each tube was transferred into a 1L flask with a growth medium containing 500 ml LB and 5 pl carbenicillin (98% (v/v)). Incubation for 48 hours in a shaking incubator at 37°C and 225 rpm to allow fiber expression was performed next. For the expression of two proteins simultaneously (referred as co-culturing), i.e., K5 and K14, the reprogrammed bacterial colonies were each loaded onto a tip of a pipette and then put into separate tubes containing 2.5ml of the antibiotic and glucose containing mixture. The tubes were then incubated for four to six hours or overnight in a shaking incubator at 37°C and 225 rpm. Then two tubes of the reprogrammed bacteria expressing the corresponding proteins were transferred into the same 1L flask containing 500ml of LB and 5 mΐ carbenicillin (98% (v/v)). The flasks were then incubated for 48 hours in a shaking incubator at 37°C and 225 rpm to allow fiber expression.

Quantitative Congo Red (CR) Amyloid Staining

Congo Red (CR) binding assay was adapted from previously published methods, see Chapman, M. R. el al. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295, 851-855 (2002).

Proteinaceous Network Formation

Two different ways of creating a novel network by the interaction of fibrin and keratin derived proteins fused onto curli were established. One is to have two reprogrammed bacteria cultures, each expressing one matching protein fused onto curli for 48 hours. Then, these two cultures are mixed together and incubated for additionally two hours to allow interaction of the matching fibrin or keratin derived proteins. The other method is to co culture two reprogrammed bacteria in the same flask for 48 hours. This way a heterogeneous expression pattern occurs of interacting proteins displayed onto curli fibers. This facilitates interaction of the matching proteins to mimic the fibrin or keratin network. Both methods, mixing and co-culturing, resulted in successful fiber aggregates, thus, to formation of the novel networks, as further illustrated and explained in the following sections and subsections. Aggregation of Engineered Curli Fibers

After 48 hours of curli fiber expression, two cultures expressing the matching proteins, i.e., K5 and K14 separately, were put together in a 2L flask. The flasks were then mixed together and placed for two hours into a shaking incubator at 37°C and 225 rpm to achieve fiber aggregation through interaction of the fused proteins onto curli. This method is referred as mixed culture. For the co-cultured bacteria expressing the matching proteins simultaneously in the same 1L flask for 48 hours, additional mixing time of two hours was not required, since the proteins were already able to interact together. The cultures of only one expressed protein, and the mixed and co-cultured bacteria were then viewed under a bright-field microscope to see and compare fiber aggregation at a magnification of 4x, lOx, 20x and 50x. Before the examination, the cells were concentrated by factor 20 and 50 for better visualization of the fibers. This was done by spinning down the culture at 6000 rpm for 10 minutes and replacement of the supernatant with DI water, i.e., for 500ml of bacterial culture the cells were resuspended in 25 ml of DI water to achieve a 20 times higher concentration. FIG.s 1A and 1B depict the two methods used for fiber aggregation.

Hydrogel Fabrication

The cultures expressing the engineered curli fibers were incubated with guanidine hydrochloride (GdmCl; Sigma- Aldrich, G4630, > 98%), at final concentration of 0.8M at 4°C for maximal one hour. After incubation, the culture was transferred onto a hydrophilic nylon net filter membrane with 11 pm pore size (Millipore Sigma) of 47 mm diameter, via vacuum filtration. The amount of transferred culture varied between 17 ml to 70 ml, depending when the filters got partially clogged. To remove living bacteria, the filter membrane was incubated with 5 ml of 8M GdmCl for 5 minutes, followed by vacuum filtration of the liquid. Next, the bacterial bio film was washed three times with 25 ml of sterile DI water. Then, the cultures on the filter membrane were treated with 5ml of 0.01% (v/v) concentration of Benzonase® nuclease (Sigma- Aldrich, 1.5 U/ml) for 5 minutes to remove DNA and RNA bound to curli fibers. Finally, the semi-purified bacteria-free fibers were incubated with 5ml of 5% (m/v) SDS in sterile water for 5 minutes. After vacuum filtration of the liquid, the filtered fibers on the membrane were washed three times with minimum 25ml DI water. The bacteria-free hydrogel was scrapped off the filter membranes and stored in small tube at 4°C. Scanning Electron Microscopy )

I. SEM of Bacterial Cultures

200 mΐ of genetically engineered bacterial culture for each functional group was vacuum filtered onto nuclepore filters (0.22pm pore size; GE Healthcare Bio-Sciences), rinsed, and fixed with 2% formaldehyde and 2% glutaraldehyde solution overnight at 4°C. Then, the samples were washed with Millipore water for 15 minutes and dehydrated with gradient steps of increasing ethanol (EtOH) concentrations (25%, 50%, 75%, 100%, 100% (v/v)). Each step takes 15 minutes of incubation time. The samples were dried with Critical Point Dryer (Autosamdri®-93l, Tousimis®). Finally, the samples were sputter coated with a platimpaladiumalloy in the ratio of 80:20. The samples were analyzed using a Zeiss

Supra55VP FE-SEM.

II. SEM of Curli Based Hydrogels

The hydrogel samples were prepared for SEM by fixing the gels with 2 m/v% glutaraldehyde and 2 m/v% paraformaldehyde in 0.1 of 0.1 M (n/V) sodium cacodylate buffer for a minimum of 2 hours at room temperature (RT). Then, the gels were gently washed with Millipore water. The solvent for every sample was gradually exchanged with increasing EtOH concentrations (25%, 50%, 75% and 100% (v/v)), with an incubation time of 15 minutes respectively. The gels were dried in a critical point dryer, placed onto SEM sample holders using silver adhesive (Electron Microscopy Sciences), and sputtered until they were coated in a 5nm layer of Pt/Pd. Imaging was performed using a Zeiss Ultra 55 Field Emission SEM.

Rheology

The mechanical properties of the different hydrogel samples were determined using a Discovery Hybrid Rheometer-3 and TRIOS software (TA Instruments, New Castle, DE). Samples were loaded between peltier plate bottom and 20mm steel top plate geometry with the gap set to be 500pm. To prevent distortion, a homogeneous mass of the hydrogels has to be ensured. During production of the gels air bubbles can occur. Removal was achieved by applying either vacuum or by centrifuging. The sample size was approximately 200m1. The excess hydrogel was trimmed along the edge of the 20mm top plate. Samples were then surrounded with mineral oil to prevent dehydration. For each hydrogel group three replicate samples were tested independently. Time sweep experiments were conducted under continuous oscillations at O.lHz with an imposed shear strain of 0.5% to measure the linear shear modulus. Example 3: Fibrous Network Expression and Hydrogel Characterization

Fiber Expression

To determine curli expression of the reprogrammed bacteria, a specific amyloid protein dye called Congo red was used. Congo red binds amyloid proteins like CsgA with high specificity. See Anne K Schiitz et al.“The amyloid-Congo red interface at atomic resolution,” Angewandte Chemie International Edition 50.26 (2011), pp. 5956-5960. Red staining indicates the production of curli and therefore confers the expression of the fused proteins onto CsgA.

Network Formation

Under a conventional bright-field microscope the ECM of reprogrammed bacteria with fibrin and keratin derived proteins were viewed and compared. The ECM of bacterial culture displaying protein a and g together (referred as CsgA-ag based ECM), through either mixing (referred as CsgA-a;/ _miXcd) (FIGs. 2C-2E) or co-culturing (referred as CsgA-ay _co - _cuitured) (FIG. 2F), showed fiber aggregation. Fibers did not bundle when only protein a (referred as CsgA-a based ECM) (FIG. 2A) or protein g (referred as CsgA-g based ECM) (FIG. 2B) was displayed on the genetically modified ECM. Similar findings were observed with reprogrammed bacterial cultures expressing keratin components. Fiber aggregation was only observed when proteins K5 and K14 were displayed together (referred as CsgA-K5Kl4 based ECM), either through mixing (referred as CsgA— KSKM _mixed ) (FIGs. 3C-3E) or co culturing (referred CsgA— K5Kl4 _co-Cuitured ) (FIG. 3F). Fibers did not aggregate when K5 (FIG. 3A) or K14 (FIG. 3B) (referred as CsgA-K5 and CsgA-Kl4 based ECM) was expressed alone.

Hydrogel Production

The production of fibrin and keratin inspired curli based hydrogels was successful. For the following sections the fibrin inspired hydrogels are labeled according to their composition and fabrication as: CsgA-a, CsgA-g, CsgA-or _mixcd CsgA-ay _to-cuiiured based hydrogel. The keratin inspired hydrogels are also categorized in: CsgA-K5, CsgA-Kl4, CsgA— K5 K 14 _mixcd CsgA-Kl5Kl4 _co-cultured based hydrogel. The production of all hydrogels was performed under the same condition. However, the quantity of gained hydrogels from 0.5 L volume of bacterial culture varied, as shown in Table 4. Table 4: Quantity of fibrin and keratin inspired curb based hydrogels quantities

The amount of gained CsgA-a based hydrogels was the lowest compared to ah other hydrogels. The amount of produced CsgA-g based hydrogels was in the same range as CsgA-K5 and CsgA-Kl4 based hydrogels. The quantity of CsgA-ay _miXCd . CsgA-ayco- cultured _? CsgA K5 K 14 _mLXC:i and CsgA Kl 5KT4 _co-cu it _U red based hydrogel were similar, but 20 to 43% less than CsgA-g, CsgA-K5 and CsgA-Kl4 respectively. Also, the appearance of the hydrogels was not the same for the different types. The hydrogels varied from transparent to more opaque.

Hydrogel Characterization

I. Hydrogel Morphology

The morphology of the produced hydrogels was examined using SEM. The images revealed a change in micro structure among the different types of fibrin and keratin inspired hydrogels. The CsgA-a based hydrogel, which represents knob“A” of the fibrin network revealed a dense pore size structure (see FIG. 4A), whereas the CsgA-g based hydrogel, which represents hole“a” of the fibrin network showed a bigger pore size structure (see FIG. 4B). However, a drastic change in the micro structure was observed in CsgA-ay _miXCd (FIG. 4C) and CsgA-ay _co-cuitured (FIG. 4D) based hydrogels. An aligned and very dense network of the fibers occurred as presented in FIGs. 4C-4D. The SEM images of keratin based hydrogels showed no difference in morphology among the different types. FIGs. 5A-5D depict the optical images of CsgA-a, CsgA-g, CsgA-ay _mixCd and CsgA-ay _co-cuitured based hydrogels, respectively. Mechanical Properties

All hydrogels were tested on a rheometer to evaluate the linear shear modulus. The hydrogels were grouped in fibrin and keratin inspired hydrogels. The quantified shear modulus for fibrin hydrogel is shown in FIG. 6.

Rheology Fibrin Inspired Hydrogels

The rheology of the fibrin inspired hydrogels revealed a significant increase in stiffness for CsgA-ay _miXed and CsgA-ay _co-cuitured compared to CsgA-a and CsgA-g based hydrogels as shown in FIG. 6. Also a difference in the shear modulus between CsgA-a and CsgA-g based hydrogels was detectable. The CsgA-g hydrogel was almost twice as stiff as the CsgA-a hydrogel. The increase in shear modulus for CsgA-ay _miXed and CsgA-ay _to-cuiiured based hydrogels was about six times compared to CsgA-a and approximately three times compared to CsgA-g hydrogels. However, the shear modulus for CsgA-ay _miXed and CsgA- ay _co-cuitured hydrogels was in the same range. The mean value for CsgA-a hydrogel was 166.49 Pa, 342.15 Pa for CsgA-g hydrogel, 955.15 Pa for CsgA-ay _miXed and 1057.67 Pa for CsgA-ayco-cuitured hydrogel.

Further tests were conducted to characterize the viscosity of the CsgA-ay _miXed and CsgA-ay _co-cuitured based hydrogels, compared to CsgA-a and CsgA-g based hydrogels, as well as CsgA-y-producing live bacteria. As shown in FIG. 8, the viscosity of CsgA-ay _miXed and CsgA— cr/ _co-cuitured based hydrogels is higher than CsgA-a and CsgA-g based hydrogels, or CsgA-y-producing live bacteria alone, demonstrating the advantageous properties of the mixed or co-cultured hydrogels for use as 3D-printing inks.

Similarly, as shown in FIGs. 9A-9D, CsgA-ay _miXed and CsgA-ay _co-cuitured based hydrogels demonstrate better rheological properties and 3D printing performances compared to CsgA-a and CsgA-g based hydrogels for use as 3D-printing inks.

Rheology Keratin inspired Hydrogels

The rheology of keratin inspired hydrogels revealed small changes in the shear modulus between its different types. The shear modulus of CsgA-K5 and CsgA-Kl4 was about the same with approximately only 10% variation. An increase in shear modulus of approximately 38% for CsgA-KSKH _mixed based hydrogels was achieved compared to CsgA- K5 and about 25% compared to CsgA-Kl4 hydrogels. The increase in shear modulus for CsgA— K5Kl4 _co-cuitured hydrogel was approximately 17% compared to CsgA-K5 and around 6% compared to CsgA-Kl4 hydrogel. However, a difference of about 17% in shear modulus between CsgA-KSKH _mixed and CsgA-K5Kl4 _co-Cuitured based hydrogel was detectable. The mean value for CsgA-K5 hydrogel was 592.37 Pa, 651.17 Pa for CsgA-Kl4 hydrogel,

817.14 Pa for CsgA-K5Kl4 _mixed hydrogel and 693.67 Pa for CsgA-K5Kl4 _co-Cuitured hydrogel.

“3D Printing” with CsgA-ag Based Hydrogel

Based on the shear thinning characteristics and to prove that fibrin inspired hydrogels can be used for future medical applications in tissue engineering, they were tested as inks for “3-dimensional (3D) printing”. To test the ability of the hydrogels in forming 3D-structures, CsgA-ag based hydrogel was loaded into a syringe and pressed through a needle. The hydrogel maintained its properties after injected through the needle and it was able build stable 3D constructs, as shown in FIG. 7. As further depicted in FIGs. 10A-10D, CsgA-ay _co - cuitured based hydrogel was built into a lO-layered circle or square, further demonstrating the ability of the hydrogel of the invention to be used as 3D-printing inks. These findings add up with the mechanical characterization of the gels with the rheological tests.

To further study the bacto-inks of the current invention for functional applications on bioactivity, sensing and binding, the bacto-inks, e.g., Bacto Prink containing CsgA-a, CsgA- g, CsgA— or _mixed or CsgA-ay _to-cuiiured based hydrogels, are further combined with functional curli fibers, or engineered living bacteria to produce functional bacto-inks for use in applications such as biosensing, signaling, binding, or other bio activities.

As depicted in FIG. 11 A, functional curli fibers were added to the cultures of

BactoPrink (containing CsgA-a, CsgA-g, CsgA-ay _miXed or CsgA-ay _to-tUiiurcd based hydrogels) and the functional inks were prepared using the protocol described in the previous sections. The resulting inks were utilized for functional applications like bio activity, sensing and binding.

As depicted in FIG. 11B, engineered living microbial cultures were incubated with BactoPrink (containing CsgA-a, CsgA-g, CsgA-ay _miXed or CsgA-ay _to-tUiiurcd based hydrogels) for 10 min and vacuum filtered to drain out excess water, which was then mixed manually to obtain a living microbial ink. Such living microbial inks were 3D printed and immersed in microbial medium having specific antibiotics and/or inducers. The living microbes in the ink were thus utilized for functional applications like secretion, bioactivity, signaling, sensing and binding.

As depicted in FIG. 11C, engineered living bacterial cultures were incubated with BactoPrink (containing CsgA-a, CsgA-g, CsgA-ay _miXed or CsgA-ay _to-tUiiurcd based hydrogels) for 10 min and vacuum filtered to drain out excess water, which was then mixed manually to obtain a living microbial ink. Such living microbial inks were 3D printed and immersed in bacterial medium having specific antibiotics and/or inducers, so as to secrete functional curli fibers in situ. The inks were then utilized for functional applications like bioactivity, secretion, signaling, sensing, binding and self-regeneration of Bacto Prink.

As depicted in FIGs. 12A and 12B, Bactoprink containing CsgA-ayco-cuitured based hydrogels was combined with living bacteria producing CsgA-a and CsgA-g and was printed into a grid pattern (FIG. 12A). Two days after the printing, the living bacteria combined with the CsgA— ay _co-cuitured based hydrogels were able to proliferate and/or produce more curli fibers (FIG. 12B), demonstrating the ability of the Bactoprink of self-regeneration.

As depicted in FIG. 13, a BactoPrink containing a CsgA-ayco-cuitured based hydrogel and live bacteria secreting an anti-cancer drug, azurin was produced and used as 3D-printing ink to print a capsule-like pattern.

As depicted in FIG. 14, a BactoPrink containing a CsgA-ayco-cuitured based hydrogel and live bacteria producing a functional curli fiber expressing a BPA (Bisphenol A) binding domain fused to the c-terminus of CsgA was produced and used as 3D-printing ink to print a spiderweb-like pattern.

Example 4: Conclusion

On the basis of the presented results, especially for mimicked fibrin, it can be concluded that described herein is a unique method to mimic fibrin and keratin. Firstly, the images on the bright-field microscope clearly indicate interaction of fibrin and keratin derived proteins when mixed or co-cultured. Secondly, for mimicked fibrin, the rheology showed a significant increase in stiffness of CsgA-ag based hydrogels suggesting that upon designed knob-hole interaction the hydrogel structure is being strengthened. Also, due to its mechanical properties the fibrin based hydrogels of ay _{m i} xed and ay _to -cuiiured function as an excellent platform for“3D printing”. Third, the interaction of a and y led to a dramatic change in the morphology of the fibrin inspired hydrogels.

Also demonstrated herein is a method of“3D printing” with fibrin inspired hydrogels, which can be used to fabricate scaffolds for wound healing in tissue engineering applications, or for drug delivery. Because existing methods for extraction and synthesis of keratin from keratin rich sources is still poor, the system disclosed herein is an ideal alternative platform to fabricate keratin based biomaterials. Those materials could be used to produce cost efficient scaffolds or coating agents to study cell behavior in cell culturing and tissue engineering applications. Moreover, because the presented system allows to display exclusively one protein or a variety of different proteins, it can be used to also personalize the scaffolds or coating materials with varying concentrations of displayed fibrin or keratin derived proteins.