CN108753183B - Underwater living body adhesive material and application thereof - Google Patents

Abstract

The invention relates to the field of underwater bonding materials, in particular to a living body underwater bonding material and application thereof. The invention provides a biological membrane which comprises TasA fusion protein, extracellular polysaccharide and BslA fusion protein. The underwater adhesive based on the bacillus subtilis biofilm provided by the invention is a living material, has the advantages of adjustable components, adjustable expression and secretion of viscous components, renewable materials and the like, and has important significance for further constructing an intelligent environment response type underwater adhesive material (such as light control, quorum sensing, odor sensing, adjustment and control and the like).

Description

Underwater living body adhesive material and application thereof

Technical Field

The invention relates to the field of underwater bonding materials, in particular to a living body underwater bonding material and application thereof.

Background

The underwater adhesive is an advanced functional material and has extremely important application in the fields of biological medicine, ocean exploration, restoration and the like. For example, in the biomedical field, suture healing of wounds, fixation of bones, and the like, adhesives are required; in the marine industry, underwater adhesives cannot be used for repairing ships, dams and sewer pipelines. Therefore, it is important to produce an underwater adhesive which is biocompatible or environmentally friendly and has high strength and high viscosity.

At present, underwater adhesives which are researched more and widely applied are mainly bionic materials designed based on the inspiration of marine organisms. The bonding characteristics of the common marine organism underwater adhesive mainly comprise: biological viscosity caused by amyloid protein structure derived from barnacle glue protein inspiration; ② biological viscosity caused by catechol (DOPA) structure derived from mytilus edulis byssus protein inspiration; ③ biological viscosity caused by the interaction of positive and negative charges derived from the inspiration of the satay worms. Through decades of development, despite great progress in the research of biomimetic underwater adhesives, these materials are often merely simulated or integrated with one or two biological inspiration underwater adhesive characteristics, and lack further integration and optimization. The adhesive for reflecting the marine organisms in the nature generally contains a plurality of adhesive protein components, and the adhesion process of the adhesive protein components is strictly regulated by the time and space of a host organism, and has the characteristics of multiple components, dynamic regulation, response to the environment and the like. Therefore, the construction of the biological inspiration underwater adhesive material with various characteristics and the realization of the intelligent underwater adhesive with each component capable of being regulated and controlled in time and space is expected to break through the bottleneck of the current adhesive technology and widen the application range of the current adhesive material in biomedical and industrial application.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a biofilm, a method of preparing the same and use thereof, which solve the problems of the prior art.

To achieve the above and other related objects, a first aspect of the present invention provides a composition comprising a TasA fusion protein comprising a TasA protein fragment and a mussel byssus protein fragment, a exopolysaccharide, a BslA fusion protein comprising a BslA protein fragment and a self-aggregating adhesive protein fragment.

In some embodiments of the invention, the TasA protein fragment is: a) polypeptide fragment with amino acid sequence shown as SEQ ID No. 39;

or, b) a polypeptide fragment having an amino acid sequence with 80% or more homology to SEQ ID NO.39 and having the function of the polypeptide fragment defined in a).

In some embodiments of the present invention, the mussel byssus protein is selected from the group consisting of perna canaliculus protein, perna viridis byssus protein, and california mussel byssus protein.

In some embodiments of the invention, the mussel byssus protein is selected from mefp-1, mgfp-2, mefp-3, mefp-5, mcfp-6, pvfp3, pvfp 5.

In some embodiments of the invention, the mussel byssus protein fragment is: c) a polypeptide fragment with an amino acid sequence shown as one of SEQ ID No. 41-47;

or d) a polypeptide fragment having an amino acid sequence which is 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homologous to one of SEQ ID Nos. 41 to 47, and having a function of the polypeptide fragment defined in c).

In some embodiments of the invention, the TasA protein is derived from bacillus subtilis.

In some embodiments of the invention, the mussel byssus protein is derived from mussel.

In some embodiments of the invention, the TasA fusion protein comprises a TasA protein fragment and a mussel byssus protein fragment in order from N-terminus to C-terminus.

In some embodiments of the invention, the weight ratio of TasA fusion protein, exopolysaccharide, and BslA fusion protein in the composition is 1-5: 80-90: 1-5.

In some embodiments of the invention, the exopolysaccharide is a polyanionic polymer.

In some embodiments of the present invention, the exopolysaccharide is a metal cation chelated exopolysaccharide, and the metal cation is selected from one or more of iron ion, magnesium ion, calcium ion and manganese ion in combination.

In some embodiments of the invention, the exopolysaccharide is derived from bacillus.

In some embodiments of the invention, the BslA protein fragment is: e) polypeptide fragment with amino acid sequence shown as SEQ ID No. 40;

or, f) a polypeptide fragment having an amino acid sequence with 80% or more homology to SEQ ID NO.40 and having the function of the polypeptide defined in e).

In some embodiments of the invention, the self-agglomerating adhesive protein is selected from the group consisting of mussel byssus protein, california, and derived adhesive short peptides.

In some embodiments of the invention, the self-agglomerating binding protein is selected from the group consisting of mcfp-3s, mcfp3 spep-spytag.

In some embodiments of the invention, the self-agglomerating adhesive protein fragment is: g) a polypeptide fragment with an amino acid sequence shown in one of SEQ ID Nos. 48-50;

or, h) a polypeptide fragment having an amino acid sequence with 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homology to one of SEQ ID Nos. 48 to 50, and having a function of the polypeptide fragment defined in g).

In some embodiments of the invention, the BslA protein is derived from Bacillus subtilis.

In some embodiments of the invention, the self-agglomerating adhesive protein is derived from mussel.

In some embodiments of the invention, the BslA fusion protein comprises, in order from N-terminus to C-terminus, a BslA fragment and a self-aggregating adhesive protein fragment.

In some embodiments of the invention, the BslA fusion protein is modified with tyrosinase.

In a second aspect, the invention provides a biofilm comprising a TasA fusion protein, an exopolysaccharide, a BslA fusion protein in a composition as described above.

In some embodiments of the invention, the amount of TasA fusion protein in the biofilm is 1-5 wt%.

In some embodiments of the invention, the content of exopolysaccharide in the biofilm is 80-90 wt%.

In some embodiments of the invention, the BslA fusion protein is present in the biofilm in an amount of 1-5 wt%.

In some embodiments of the invention, the biofilm is derived from bacillus.

In a third aspect, the present invention provides an expression system capable of forming a biofilm as described above.

In some embodiments of the invention, the expression system comprises an engineered bacterium.

In some embodiments of the invention, at least a portion of the engineered bacteria comprise a construct capable of expressing the TasA fusion protein, or have an exogenous polynucleotide encoding the TasA fusion protein integrated into the genome.

In some embodiments of the invention, at least a portion of the engineered bacteria contain a construct capable of expressing the BslA fusion protein, or have an exogenous polynucleotide encoding the BslA fusion protein integrated into the genome.

In some embodiments of the invention, at least a portion of the engineered bacteria are capable of producing EPS polysaccharides, at least a portion of the engineered bacteria comprising a construct capable of expressing tyrosine, or having an exogenous polynucleotide encoding tyrosine integrated into the genome.

In some embodiments of the invention, the engineered bacterium is selected from bacillus.

The fourth aspect of the present invention provides a method for preparing the biofilm, comprising: culturing said expression system under conditions suitable for the formation of said biofilm.

In a fifth aspect, the present invention provides a biofilm expressed by the expression system as described above or by the production method as described above.

In a sixth aspect the present invention provides the use of a composition as described above, a biofilm as described above in the field of underwater adhesive preparation.

Drawings

FIG. 1 shows an illustration of the biofilm-associated genes of different Bacillus subtilis strains constructed according to the invention;

FIG. 2 shows a map of an expression vector of the present invention based on pHT01 (a: pHT 01-tapA-sipW-tasA; b: pHT01-tapA-sipW-tasA-mefp 5; c: pHT 01-bsLA; d: pHT01-bsLA-3 sp-spytag);

FIG. 3 shows TEM images of nanofibers of the present invention (a: TD; b: TD/pHT 01-tapA-sipW-tasA; c: TD/pHT01-tapA-sipW-tasA-mefp 5);

FIG. 4 shows the rheological test curves of the biofilm (TD series) of the present invention (a: amplitude oscillation test mode results; b: frequency oscillation test mode results);

FIG. 5 shows the lap shear viscometric test curves (a: schematic view of lap shear test apparatus; b: graph of the viscometric test results) of the biofilms (TD series) according to the present invention;

FIG. 6 shows the rheological test curves of the biofilms (TD and DD) according to the present invention (a: amplitude oscillation test mode result; b: frequency oscillation test mode result);

FIG. 7 shows the IR spectrum of the biofilm (TD and DD) of the present invention after mixing with iron ions;

FIG. 8 shows a scanning electron microscope image of the biofilm (DD) of the present invention after mixing with iron ions;

FIG. 9 shows the results of the lap shear adhesion test of the biofilm (DD) of the present invention and its iron ions;

FIG. 10 shows fluorescence microscopy images of the invention detecting spytag interaction with mcherry-spycatcher;

FIG. 11 shows the rheological test curves of the biofilm (Db series) of the present invention (a: amplitude oscillation test mode results; b: frequency oscillation test mode results);

FIG. 12 shows the lap shear viscometric test curves (a: schematic view of lap shear test apparatus; b: graph of the viscometric test results) of the biofilm (Db series) of the present invention;

FIG. 13 shows the construction and map of tyrosinase expression vector according to the present invention;

FIG. 14 shows a rheological test curve for an integrated biofilm of the present invention;

FIG. 15 shows the final lap shear tack test results for integrated biofilms of the invention;

FIG. 16 shows the lap shear tack test results of the integrated biofilms of the invention under different environments;

FIG. 17 shows the results of lap shear viscometric testing of integrated biofilms of the invention after subculture regeneration;

FIG. 18 shows the injectability verification of the integrated biofilms of the invention;

FIG. 19 shows an example of the adhesive application of the integrated biofilm of the present invention on different substrate materials.

Detailed Description

The inventor of the invention has completed the invention on the basis of a large number of exploratory experiments, by taking the components of the bacillus subtilis biofilm as a basic frame and relying on TasA (amyloid protein) fusion protein, Exopolysaccharides (EPS) and BslA (surface hydrophobin) fusion protein, the purposes of adjusting the adhesive components of the living adhesive material and gradually optimizing the adhesive strength are realized.

In one aspect of the invention, a composition can include a TasA fusion protein that can include a TasA protein fragment and a mussel byssus protein fragment. The TasA protein fragment may be: a) the polypeptide fragment with the amino acid sequence shown as SEQ ID No.39, can also be b) polypeptide fragments with the amino acid sequence having homology of more than 80%, more than 85%, more than 90%, more than 93%, more than 95%, more than 97%, or more than 99% with the SEQ ID No.39 and the functions of the polypeptide fragments defined by a), wherein the amino acid sequence in b) specifically refers to: the amino acid sequence shown as SEQ ID No.39 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids at the N-terminal and/or C-terminal, and the encoded polypeptide fragment has the amino acid sequence of the function of the polypeptide fragment encoded by the amino acid sequence shown as SEQ ID No. 39. The mussel foot silk protein (mussel foot proteins) may be selected from, but not limited to, mussel foot silk protein (mefp), Perna viridis foot silk protein (pvfp), california mussel foot silk protein (Mytilus californicanus foot protein), mediterranean mussel foot silk protein (Mytilus galloprovincialis foot protein), etc., and more specifically may be selected from, but not limited to, mefp-1, mgfp-2, mefp3, mefp5, mcfp-6, pvfp3, pvfp5 or variants thereof, etc., for example, the mussel foot silk protein fragment may be: c) the polypeptide fragment with an amino acid sequence shown in one of SEQ ID No.41-47, or d) a polypeptide fragment with an amino acid sequence which has more than 80%, more than 85%, more than 90%, more than 93%, more than 95%, more than 97%, or more than 99% homology with one of SEQ ID No.41-47 and has the function of the polypeptide fragment defined by c), wherein the amino acid sequence in d) specifically refers to: the polypeptide fragment encoded by the polypeptide fragment has an amino acid sequence of the polypeptide fragment encoded by the amino acid sequence shown in one of SEQ ID Nos. 41 to 47, which is obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or1 to 3) amino acids to the amino acid sequence shown in one of SEQ ID Nos. 41 to 47, or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or1 to 3) amino acids to the N-terminal and/or C-terminal. The TasA protein is typically derived from Bacillus subtilis (Latin name) and the Mytilus byssus protein is typically derived from Mytilus edulis (Latin name: Mytilidae). In a specific embodiment of the invention, the TasA fusion protein comprises a TasA protein fragment and a mussel byssus protein fragment in sequence from N-terminus to C-terminus.

The composition provided by the invention can also comprise exopolysaccharides. The exopolysaccharide is typically a polyanionic polymer, typically one of the major components of a biofilm, which is typically a high molecular substance secreted by a microorganism, and more particularly may be a natural high molecular polymer, and the microorganism secreting the exopolysaccharide (i.e., the source of the exopolysaccharide) may be, including but not limited to, bacillus (e.g., bacillus subtilis), pseudomonas aeruginosa, bacillus aceticus, escherichia coli, and the like. The exopolysaccharide may be chelated with a metal cation, and in the composition, the exopolysaccharide may also be a metal cation-chelated exopolysaccharide. What is needed isThe metal cation may be a combination including, but not limited to, one or more of iron ion, magnesium ion, manganese ion, calcium ion, copper ion, sodium ion, etc., and in one embodiment of the present invention, the metal cation is Fe³⁺Ions. Methods of chelating exopolysaccharides to metal cations will be known to those skilled in the art, for example, exopolysaccharides can be constructed from exopolysaccharides that are chelated to Fe by introducing a metal cation (e.g., a salt containing a metal cation) into the composition to chelate the exopolysaccharide to the metal cation³⁺By means of which an increase in viscosity is obtained

The BslA fusion protein can also be included in the composition provided by the invention, and the BslA fusion protein can include a BslA protein fragment and a self-aggregating adhesive protein fragment. The BslA protein fragment may be: e) the polypeptide fragment with the amino acid sequence shown as SEQ ID No.40 can also be f) polypeptide fragments with the amino acid sequence having homology of more than 80%, more than 85%, more than 90%, more than 93%, more than 95%, more than 97%, or more than 99% with the SEQ ID No.40 and the functions of the polypeptide fragment defined by e), wherein the amino acid sequence in f) specifically refers to: the amino acid sequence shown as SEQ ID No.40 is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids at the N-terminal and/or C-terminal, and the encoded polypeptide fragment has the amino acid sequence of the function of the polypeptide fragment encoded by the amino acid sequence shown as SEQ ID No. 40. The self-agglomerating adhesive protein generally refers to mussel byssus protein capable of self-agglomerating, and can be generally Mytilus galloprovincialis byssus protein capable of self-agglomerating, derived adhesive short peptides and the like, and more particularly can include but is not limited to mcfp-3s, mcfp3 spen (also referred to herein as 3sp for short), mcfp3 spen-spytag and the like. For example, the self-agglomerating adhesive protein fragment may be: g) the polypeptide fragment with an amino acid sequence shown in one of SEQ ID No.48-50, or h) the polypeptide fragment with an amino acid sequence which has homology of more than 80%, more than 85%, more than 90%, more than 93%, more than 95%, more than 97%, or more than 99% with one of SEQ ID No.48-50 and has the function of the polypeptide fragment defined by c), wherein the amino acid sequence in d) specifically refers to: the polypeptide is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids to the amino acid sequence shown in one of SEQ ID Nos. 48-50, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids to the N-terminal and/or C-terminal of the polypeptide, and the encoded polypeptide has the functional amino acid sequence of the polypeptide encoded by the amino acid sequence shown in one of SEQ ID Nos. 48-50. The BslA protein is typically derived from Bacillus subtilis (Latin name) and the self-agglomerating adhesive protein is typically derived from mussel (Latin name: Mytilidae). In one embodiment of the present invention, the BslA protein fusion protein comprises a BslA protein fragment and a self-aggregating adhesive protein fragment in order from the N-terminus to the C-terminus. The BslA fusion protein is typically modified with tyrosinase (tyrosinase), which typically means that the tyrosine moiety in the BslA fusion protein is modified to DOPA, so that its contribution to viscosity can be further increased.

In the composition provided by the invention, the mass ratio of the TasA fusion protein to the extracellular polysaccharide can be usually 0.1-10: 60-90, or 1-5: 80-90, the mass ratio of the TasA fusion protein to the BslA fusion protein can be usually 0.1-10: 0.1-10, or 1-5: 1-5. The preparation method of the composition should be known to those skilled in the art, and for example, the above fusion protein and/or exopolysaccharide may be prepared, purified, and mixed by chemical synthesis, microbial culture, etc. to obtain the above composition.

In another aspect of the invention, there is provided a biofilm which may comprise a TasA fusion protein as described above. The content of the TasA fusion protein in the biofilm can be 0.1-10 wt%, or 1-5 wt%.

The biological membrane provided by the invention can also comprise the exopolysaccharide. In the biological membrane, the content of the extracellular polysaccharide is 60-90 wt%, or 80-90 wt%.

The biological membrane provided by the invention can also comprise the BslA fusion protein. In the biological membrane, the content of BslA protein fusion protein can be 0.1-10 wt%, or 1-5 wt%.

In the biofilm provided by the present invention, the biofilm is generally derived from Bacillus (latin name: Bacillus), and specifically can be, for example, Bacillus subtilis (latin name: Bacillus subtilis), Bacillus megaterium (latin name: Bacillus megatherium), Bacillus cereus (latin name: Bacillus cereus), that is, the biofilm can be generally produced by the above-mentioned bacteria, and the strain can express and secrete relevant fusion protein and polysaccharide, so as to produce the biofilm as described above. For example, since Bacillus subtilis belongs to gram-positive bacteria and has a strong ability to extracellularly secrete proteins, the fusion adhesive protein is expressed and secreted in an expression strain, and the secreted proteins can self-assemble extracellularly to form adhesive fibers.

In another aspect, the present invention provides an expression system capable of forming a biofilm as described above.

In the expression system provided by the invention, the expression system can be engineering bacteria. In the expression system, the TasA fusion protein, the extracellular polysaccharide and the BslA fusion protein can be simultaneously expressed in the same engineered bacterium (group), for example, the engineered bacterium can simultaneously contain a construct capable of expressing the TasA fusion protein and the BslA fusion protein, or a polynucleotide encoding the TasA fusion protein and the BslA fusion protein which are exogenously integrated in the genome, and the engineered bacterium can be bacillus (for example, bacillus subtilis) and the like. In the expression system, the TasA fusion protein, the exopolysaccharide and the BslA fusion protein can be expressed in a plurality of different engineering bacteria (groups), for example, at least part of the engineering bacteria can express the TasA fusion protein, and for example, at least part of the engineering bacteria can contain a construct capable of expressing the TasA fusion protein or a polynucleotide encoding the TasA fusion protein with exogenous genes integrated in the genome; for another example, at least a portion of the engineered bacteria may express exopolysaccharides as described above, and for another example, the engineered bacteria may be bacillus (e.g., bacillus subtilis) or the like; for another example, at least a portion of the engineered bacteria can express a BslA fusion protein as described above, and for another example, at least a portion of the engineered bacteria comprise a construct capable of expressing the BslA fusion protein or have a polynucleotide encoding the BslA fusion protein integrated into its genome that is foreign. One skilled in the art can select suitable engineered bacteria, constructs, and the like, which can be, for example, bacillus generally, and more particularly, combinations comprising one or more of but not limited to bacillus subtilis, bacillus megaterium, bacillus cereus, and the like, and construct the expression system using suitable methods, which can be, for example, combinations comprising one or more of but not limited to, a pHT01 vector, a pHT43 vector, a pHT254 vector, a pMAD vector, and the like.

In the expression system provided by the invention, at least part of the engineering bacteria can also express tyrosinase, for example, at least part of the engineering bacteria contains a construct capable of expressing tyrosinase or integrates exogenous polynucleotide for encoding tyrosinase in the genome. The expressed tyrosinase can be used to modify the BslA fusion protein so that its contribution to viscosity can be further increased.

In another aspect, the present invention provides a method for preparing the biofilm, comprising: culturing an expression system as described above under conditions suitable for the formation of said biofilm. The conditions for forming a biofilm generally depend on the kind of expression system used, and suitable conditions for forming a biofilm should be known to those skilled in the art, and for example, a method of culturing a biofilm using solid MSGG or MSGG liquid may be used.

In another aspect of the present invention, there is provided a biofilm expressed by the expression system as described above or the production method as described above.

The invention also provides application of the biological membrane in the field of preparation of underwater adhesives. The underwater adhesive may be an adhesive for various substrate materials, for example, may be various suitable inorganic substances including, but not limited to, one or more combinations of inorganic glass, metal, paper material, ceramic material, bone, artificial bone, etc., and/or organic substances including, but not limited to, one or more combinations of organic glass, textile, plastic, polyurethane, polymeric material such as PDMS, cloth, woven material such as nylon, etc.

The underwater adhesive based on the bacillus subtilis biofilm provided by the invention is a living material, has the advantages of adjustable components, adjustable expression and secretion of viscous components, renewable materials and the like, and has important significance for further constructing an intelligent environment response type underwater adhesive material (such as light control, quorum sensing, odor sensing, adjustment and control and the like). Compared with the prior art, the main technical advantages and characteristics of the invention include the following aspects:

1) the finally obtained adhesive material has basic characteristics of living bodies (living cells), including excellent performances of biological regeneration, environmental response, programmability, self-repair and the like, which are not possessed by common macromolecular and pure protein adhesive materials.

2) The living body adhesive material is based on a genetically modified bacillus subtilis biofilm, and adhesive materials with different molecular characteristics are designed by using a gene module method on the basis, and the adhesive materials show different adhesive strength characteristics; finally, the gene engineering designs and integrates the adhesive material containing various adhesive molecular characteristics on the genome to obtain the optimized living adhesive material.

3) The integrated living body adhesive material obtained through rational design has the characteristics of strong environmental tolerance, injectability and the like, and shows good underwater viscosity to common material substrates.

4) The living adhesive material is constructed based on genetically modified bacillus subtilis, and other functional groups or proteins (including functional groups related to adhesion and non-adhesion) can be introduced to construct the multifunctional living adhesive material.

5) Since the biological membrane is directly related to living cells, the technology can realize controllable expression and secretion of the functional fusion protein on time space by using the perception of bacteria to the external environment (such as pH, illumination, temperature, smell and the like) or the expression regulation of bacteria by gene loops (such as chemical induction, quorum sensing and the like), thereby realizing the intelligent concept of the bonded living material.

6) Compared with Escherichia coli, the Bacillus subtilis belongs to Generally Recognized Safe (GRAS) microorganisms, has better biological safety, and is expected to have important application potential in the fields of biological medicine and other industries.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

The specific construction method of the engineering bacteria used in the present embodiment is as follows:

the bacillus subtilis biological membrane mainly comprises the following components: extracellular polysaccharide, amyloid protein and surface hydrophobin are respectively expressed by an epsA-O gene cluster, a tasA gene and a bsLA gene in a coding manner, and a strain TD with all the biomembrane related genes knocked out is constructed for the expression of a tasA-R fusion protein in a modular design. EPS and Fe for Modular design³⁺Constructing the strain DD with the preserved eps gene of the biomembrane. Constructing a bacterial strain Db with a knockout biomembrane bslA gene for modularly designing the expression of the bslA-R' interfacial viscous fusion protein.

The three different Bacillus subtilis strains can be obtained by gradually knocking out the genes bsLA, tasA and epsA-O. Specifically, based on a complete genome sequenced Bacillus subtilis 168 genome sequence (genome sequencing genebank No. NC-000964), upstream specific primers, upstream-F and upstream-R, and downstream specific primers, downstream-F and downstream-R, are designed. The gene group of wild Bacillus subtilis 3610 is used as a template, the upstream and downstream homologous sequences of a target knockout gene on the gene group are amplified by PCR (polymerase chain reaction) and about 1kb, pMAD is used as an integrated plasmid vector, and related molecular biological experimental methods such as plasmid construction, strain transformation, strain construction and the like are the same as those in the patent (201611156490. X).

The difference is that for constructing Db strain, the upstream and downstream fragment primers required for amplifying and deleting bslA are respectively D bslA upstream-F (SEQ ID NO: 1), D bslA upstream-R (SEQ ID NO: 2), D bslA downstream-F (SEQ ID NO: 3) and D bslA downstream-R (SEQ ID NO: 4), and the used restriction enzyme cutting sites of the integration vector are SmaI and EcoRI. On the basis, the DD strain is further constructed, the upstream and downstream segment primers required by amplifying and deleting the tasA are respectively D tasA upstream-F (SEQ ID NO: 5), D tasA upstream-R (SEQ ID NO: 6), D tasA downstream-F (SEQ ID NO: 7) and D tasA downstream-R (SEQ ID NO: 8), and the enzyme cutting sites of the used integration vector are SmaI and NcoI. On the basis, a TD strain is further constructed, and primers of an upstream fragment and a downstream fragment required for amplifying and deleting epsA-O are respectively D eps upstream-F (SEQ ID NO: 9), D eps upstream-R (SEQ ID NO: 10), D eps downstream-F (SEQ ID NO: 11) and D eps downstream-R (SEQ ID NO: 12). The restriction sites of the used integration vectors were BamHI and SalI and, for constructing the integration strain T5B3, the gene cluster of tasA-sipW-tasA-mefp 5 (SEQ ID NO: 14) was synthesized by the company and the primers thereof were tasA-mefp5-F (SEQ ID NO: 25) and tasA-mefp5-R (SEQ ID NO: 26), and the upstream and downstream amplification primers of tasA-mefp5upstream-F (SEQ ID NO: 23), tasA-mefp5upstream-R (SEQ ID NO: 24) and tasA-mefp5downstream-F (SEQ ID NO: 27) and tasA-mefp5downstream-R (SEQ ID NO: 28) were designed, and the restriction sites of the used integration vectors were SmaI and NcoI. On this basis, when bsLA-3sp (3sp is mfp3sp for short, the same shall apply hereinafter) is introduced into the genome, the same company synthesizes bsLA-3sp-spytag (SEQ ID NO: 19) and designs primers of bsLA-3sp-F (SEQ ID NO: 31) and bsLA-3sp-R (SEQ ID NO: 32), and designs upstream and downstream amplification primers of bsLA-3sp upstream-F (SEQ ID NO: 29), bsLA-3sp upstream-R (SEQ ID NO: 30) and bsLA-3sp downstream-F (SEQ ID NO: 33 and bsLA-3sp downstream-R (SEQ ID NO: 34), and the cleavage sites of the integrated vectors are SmaI and NcoI.

All the tapA-sipW-tasA-R, bslA-R' and tyrosinase related genes are obtained by total synthesis of the company, the used expression vector is pHT01(mobitec company), and enzyme cutting sites are BamHI and SmaI. The specific method for constructing an expression vector containing a target gene is the same as that disclosed in the patent (201611156490. X). Specifically, for the construction of the control vector pHT01-tapA-sipW-tasA, the gene sequence of tapA-sipW-tasA was SEO ID NO.13, and the upstream and downstream primers were TO-F (SEQ ID NO: 15) and TO-R (SEQ ID NO: 16), respectively. For the construction of pHT01-tapA-sipW-tasA-mefp5, the upstream and downstream primers were TO-F (SEQ ID NO: 15) and TO-mefp5-R (SEQ ID NO: 17), respectively, for the construction of the control vector pHT01-bsLA, the gene sequence of bsLA was SEO ID NO.18, and the upstream and downstream primers were bsLA-F (SEQ ID NO: 20) and bsLA-R (SEQ ID NO: 21), respectively. For the construction of pHT01-bsLA-3sp-spytag, fragment bsLA-3sp-spytag (SEQ ID NO: 19) was synthesized by the company as a whole, and the upstream and downstream primers were bsLA-F (SEQ ID NO: 20) and bsLA-3sp-spytag-R (SEQ ID NO: 22), respectively. For the construction of pHT01-tyrosinase, the fragment tyrosinase (SEQ ID NO: 36) was synthesized by the company as a whole, and the upstream and downstream primers were tyrosinase-F (SEQ ID NO: 37) and tyrosinase-R (SEQ ID NO: 38), respectively.

In addition to the above genetic engineering procedures, the LB medium, biofilm medium MSGG, and liquid biofilm culturing methods referred to in the present invention are equivalent to those described in the patent No. 201611156490. X. It should be noted that the solid LB or MSGG culture medium is prepared by adding 1.5% agar powder on the basis of liquid culture medium, and the method for culturing the biological membrane on the solid culture medium comprises the following steps: the single colonies on the overnight streaked plates were inoculated in 5mL of LB medium at 37 ℃ for 3 hours with shaking at 220rpm, and again inoculated in 5mL of LB medium at 1% ratio at 37 ℃ for 3 hours with shaking at 220 rpm. The cells were collected by centrifugation at 5000g, resuspended at OD 0.8-1.0 with deionized water, and pipetted at 2.5. mu.L onto MSGG solid plates. If the host bacteria contain pHT01 expression vectors, the culture medium contains 5 mug/mL chloramphenicol in the inoculation process; for the strain containing pHT 1-bslA-R' vector, the culture medium for finally culturing the biological membrane needs to contain 1mmol/L IPTG; for the strain containing pHT1-tyrosinase vector, 0.4. mu.g/mL of CuSO was required in the medium for final culture of biofilm₄；

In addition, the immunoassay method in Transmission Electron Microscopy (TEM) as referred to in the present invention is the same as that described in the patent (201611156490. X).

Example 1: modular design based on amyloid TasA and mussel byssus protein viscosity characteristics

In this example, the strain TD (fig. 1d) with the complete knockout of the gene of the bacillus subtilis biofilm is used, so that the influence of the self-viscosity of the biofilm on the modular design can be reduced to the maximum extent, and the contribution of the design to the improvement of the biofilm viscosity can be reflected more effectively. The invention constructs foreign pHT01-tapA-sipW-tasA plasmid for expressing amyloid TasA as a control (figure 2a) and pHT01-tapA-sipW-tasA-mefp5 plasmid for simultaneously expressing amyloid and mussel byssus protein (figure 2 a).

Activated E.coli TG1(Biovecter, Biovector 105812-6) was picked up from LB plate, inoculated into 5ml of LB medium, and cultured overnight with shaking at 37 ℃. Inoculating the bacterial liquid at a ratio of 1:100, and culturing 250 μ l of the bacterial liquid in 25ml of LB culture medium at 37 ℃ for 2-3 hours under shaking until OD600 is about 0.5. The bacterial liquid is transferred into a 50ml centrifuge tube and placed on ice for 10 min. Centrifuging at 4000r/min for 10min at 4 deg.C, discarding supernatant, and inverting the tube for 1min until the liquid is drained. The cells were gently suspended in 10ml of 0.1mol/L CaCl2 solution precooled on ice and allowed to stand on ice for 30 min. Centrifuging at 0-4 deg.C and 4000r/min for 10min, discarding supernatant, adding 2ml precooled 0.1mol/L CaCl2 solution, suspending cells gently, and standing on ice to obtain competent cells. 200 μ l of the prepared competent cells are taken, 1 μ g of the integrated plasmid constructed in the step is added, and the cells are transformed by a chemical transformation method. The plasmid was extracted from Escherichia coli TG1 obtained by the method described in the previous discussion of competence using a plasmid-upgrading kit (Tiangen, DP103), and transformed into a strain of Bacillus subtilis to be knocked out by Spizzen transformation. The specific steps of the Spizisen transformation method are as follows: bacillus subtilis 3610 (Bacillus Genetic Stock Center (BGSC), 1L26) to be transformed is activated. Fresh single clones were selected and inoculated into 3ml of 2XYT medium and cultured overnight with shaking at 37 ℃. The strain is inoculated in 5ml of medium A according to the proportion of 1:100, and the shake culture is carried out for 3.5h at the temperature of 37 ℃. Inoculating the bacteria in medium A into medium B according to the ratio of 1:10, and performing shake culture at 37 ℃ for 1.5h to obtain competent cells. At this time, competent cells were prepared, and 1. mu.g of the integrated plasmid DNA extracted from Escherichia coli TG1 was added to 500. mu.l of the competent cells. Standing in water bath at 37 deg.C for 60 min. Culturing competent cells at 37 deg.C for 2h by shaking, spreading on a plate with chloramphenicol resistance gene, and standing in a constant temperature incubator at 37 deg.C for 8 h. The strain preserved at the temperature of minus 80 ℃ is inoculated into LB culture medium for overnight culture, the thalli are collected by centrifugation, suspended by deionized water until OD600 is about 1.0, added into 4ml of MSGG liquid culture medium according to the proportion of 1 percent, and kept still for two days. 10 μ l of biofilm solution cultured in liquid MSGG culture medium for 2 days was dropped on a copper mesh (BZ 10024a) and allowed to stand for 2-5min, and then blotted with filter paper. Take 10. mu.l of blocking buffer on a copper net, stand for 30min, and suck to dry. The blocking buffer formula is as follows: to 200ml of PBS (life technology, 00051) were added 40. mu.l of Tween 20 (raw, A600560-0500) and 0.4g of skim milk powder (BD Difco, 232100). Mu.l of primary antibody solution (used after the primary antibody is diluted 150 times to the blocking buffer) is dropped on a copper net, and the mixture is kept stand for 2h and sucked dry. Wash 10. mu.l of PBST once and blot dry. The formulation of PBST is: PBS + 0.1% Tween 20. Mu.l of secondary antibody solution (used after 50-fold dilution of secondary antibody to PBST) was dropped onto the copper mesh for 1h and blotted dry. Wash 10. mu.l of PBST once and blot dry. Washed once more with 10. mu.l of deionized water and blotted dry. Mu.l (2.5mg/ml) of uranium acetate solution was dropped on a copper mesh, stained for 30s, and blotted dry. It can be observed that the strain TD with the biofilm gene completely knocked out does not contain any nanofiber structure, while the strain TD carrying plasmids pHT01-tapA-sipW-tasA and pHT01-tapA-sipW-tasA-mefp5 has similar amyloid fiber structure, thereby indicating that the fusion protein TasA-mefp5 can be correctly expressed, secreted and formed fibers in the Bacillus subtilis strain.

The method for culturing the biofilm by using the solid MSGG is adopted, and the viscoelasticity of the biofilm is measured by using a rheometer (Antopa MCR 101). Cultured TD, TD/pHT01-tapA-sipW-tasA (TD strain carrying pHT01-tapA-sipW-tasA plasmid) and TD/pHT01-tapA-sipW-tasA-mefp5 (TD strain carrying pHT01-tapA-sipW-tasA-mefp5 plasmid) biofilms are scraped off from MSSG solid culture medium respectively and are uniformly coated on a rheometer rotor cp 25-2. The change of Storage modulus (Storage modulus) to strain (gamma) was measured (fig. 4a), wherein the oscillation frequency of the measurement parameter was fixed at 10rad/s and the strain amplitude was 0.01% -100%, and it can be seen that the expression of the fusion protein TasA-mefp5 effectively improved the elasticity and toughness of the TD strain. And simultaneously measuring the change of the storage modulus to the frequency (omega) (figure 4b), wherein the strain amplitude of the measurement parameter is fixed to be 0.1%, and the oscillation frequency amplitude is 0.1-100rad/s, so that the expression of the fusion protein TasA-mefp5 under high frequency and low frequency can effectively improve the elastic modulus of the TD strain and promote the internal cross-linked network structure.

The method for culturing the biofilm by using the solid MSGG is adopted, and the shear viscosity strength of the biofilm is measured by using an universal mechanical device (Instron). The cultured biological membranes are respectively scraped from the MSSG solid culture medium and are evenly coated between two smooth stainless steel skins. The stainless steel plate had a size of 5cm long, 1cm wide and 0.2mm thick, and the area of the plate was a square area of 1cm long and 1cm wide (FIG. 5 a). The test samples were incubated in a 30 ℃ incubator (DHP-9032, Shanghai-Heng scientific instruments Co., Ltd.) for 2 hours and then subjected to tensile testing. The stretching speed of the universal mechanical device is set to be 5mm/min, and the distance between the upper stretching clamp and the lower stretching clamp is 5 cm. In addition, for the biofilm of TD/pHT01-tapA-sipW-tasA-mefp5, tyrosine rich in the fusion protein TasA-mefp5 was converted into catechol (DOPA) with excellent viscosity by adding 2.5. mu.L of tyrosinase at an additional 1mmol/L, and the shear viscosity thereof after modification was tested (FIG. 5 b). The result shows that the biomembrane which is not treated by the tyrosinase and contains the fusion protein TasA-mefp5 has higher viscosity compared with TD, and the viscosity of the biomembrane is greatly improved after being treated by the tyrosinase and reaches 63.47 +/-9.75 kPa. This indicates that the fusion of mussel byssus protein and amyloid TasA does contribute significantly to the biofilm viscosity.

In conclusion, the mussel byssus protein with excellent underwater adhesion performance is fused with the amyloid protein fusion TasA which plays a role of a skeleton in the biological membrane, so that the viscoelasticity, toughness and shear viscosity of the biological membrane can be improved, the structure of the biological membrane is more compact, and the internal viscosity and the interfacial viscosity are greatly enhanced.

Example 2: based on EPS and Fe³⁺Electrostatic interaction mediated viscous character modular design

EPS polysaccharide, which is a major component of biological membranes, is generally known as a polyanionic polymer by introducing transition metal ions (Fe)³⁺) Chelating with it can further improve its internal crosslinking characteristics. This example uses TD strain as a control, DD strain with EPS component (FIG. 1c), and Fe by comparing TD and DD biofilms³⁺Determination of EPS and Fe³⁺And measuring its effect on the viscosity of the biofilm.

The method for culturing the biofilm by using the solid MSGG is adopted, and the viscoelasticity of the biofilm is measured by using a rheometer (Antopa MCR 101). The measurement method and parameters were the same as in example 1 above. It can be seen that the polysaccharide EPS component is produced, which, although not greatly increasing the elasticity of the biofilm, greatly enhances the toughness of the biofilm (fig. 6 a). Furthermore, at low frequencies TD almost coincides with the storage modulus of DD, while at high frequencies DD is about twice the TD modulus, indicating that EPS effectively increases the physical cross-linked structure of the biofilm (fig. 6 b).

Method for culturing biological membrane by adopting solid MSGG and identifying EPS and Fe by using Fourier transform infrared spectroscopy (FTIR, Perkinelmer)³⁺The interaction of (a). Respectively scraping the cultured TD and DD biological membranes into a 50mL centrifuge tube, adding distilled water to fully resuspend the biological membranes, dividing the solution into two parts, wherein one part only contains the biological membrane suspension, and the other part is added with 0.1mL of 1mol/L ferric chloride solution. Both TD and DD samples were lyophilized in a lyophilizer (laboco) for 24 hours. After freeze-drying, the samples were assayed with a measurement parameter spectral range of 900cm^-1—1800cm^-1(FIG. 7). It can be seen that TD and DD add Fe³⁺At 1084cm^-1And 979cm^-1Obvious difference in absorption indicates that EPS and Fe³⁺The main functional group for interaction is phosphate PO₃ ^2-(ii) a Furthermore, the infrared absorption spectra of TD and DD are 1598cm^-1There is also a significant change in the position indicating the presence of a COO moiety in the biofilm composition^-With Fe³⁺The interaction of (a).

Method for culturing biological membrane by adopting solid MSGG and using scanning electron microscopeObservation of EPS and Fe with mirror (SEM, JSM 7800F)³⁺The difference in morphology that results from the interaction of (a). Taking a DD biomembrane colony cultured on an MSGG culture medium, adding 1mol/L ferric chloride solution, coating the colony on clean aluminum foil paper, fixing the colony for 1 hour by using 5% glutaraldehyde solution (Annaiji), and then performing gradient dehydration by using ethanol (the ethanol concentration is 50%, 60%, 70%, 80%, 90% and 100% in sequence). The prepared sample was subjected to sputtering for 30 seconds in a metal spraying machine and then observed by SEM (FIG. 8, scale bar 1 μm). As can be seen, addition of Fe³⁺The distance between the biofilm matrix and the bacteria is then significantly tighter, which also suggests that electrostatic interactions play a critical role in the dehydrating polycondensation of the biofilm internal structure.

The method for culturing biofilms using the above solid MSGG and measuring the shear viscosity strength of biofilms using an universal mechanical apparatus (Instron) (fig. 9). The measurement method and parameter settings were the same as in example 1. As can be seen, addition of Fe³⁺After that, the shear viscosity of the biomembrane material is greatly improved by about 4 times to 63.8 +/-8.01 kPa.

In summary, a biofilm containing a polysaccharide component will have a significant amount of phosphate groups PO₃ ^2-The polyanion not only serves as a skeleton to improve the toughness of the biological membrane, but also can generate obvious electrostatic interaction with metal cations, thereby further improving the intrinsic coagulation of the biological membrane and finally showing that the viscosity of the biological membrane is obviously enhanced.

Example 3: modularized design based on surface hydrophobin BslA fusion viscous short peptide

BslA protein is used as a hydrophobic protein on the surface of a biological membrane of bacillus subtilis and has the advantage of natural engineered interfacial adhesion. This example is based on Db strain and introduces pHT01-bsLA-3sp-spytag plasmid (FIG. 2d) to obtain expression of the fusion protein bsLA-mefp3s-spytag at the interface of the biological membrane, while pHT01-bsLA plasmid (FIG. 2c) is used as control and the transfection method of the plasmid is described in example 1. The functional modification of the bslA gene is proved to be successful through the interaction of Spytag and mCherry-spycatcher (the same as the patent), and the influence of the fusion BslA-3sp-Spytag on the viscosity of the biological membrane is identified by adopting a rheological test and a universal mechanical device tensile test.

The method for culturing the biological membrane by adopting the liquid MSGG is used for culturing the Db/pHT01-bsLA-3sp-spycatcher biological membrane. Sucking 500 mu L of biomembrane bacteria liquid into a 1.5mL EP tube, centrifuging for 1min at 10000g, discarding the supernatant, adding 200 mu L of mCherry-spycatcher solution, combining for 40min on a rotary shaking table (QB-208, Linbel), centrifuging for 1min at 10000g, discarding the supernatant, picking out a proper amount of centrifuged precipitate, coating the precipitate on the center of a glass slide, dripping 20 mu L of deionized water, and covering the glass slide. For the control group, 200. mu.L of deionized water was added directly without mCherry-spycatcher and combined on a rotary shaker for 40min, and the rest was as above. Observing the sample under a fluorescence microscope (Leica) (FIG. 10, scale bar 20 μm), it can be seen that the B.subtilis biofilm carrying the fusion plasmid pHT01-bsLA-3sp-spytag was able to bind to mCherry-spycacher and develop red color, so that the surface BslA-3sp-spytag was able to express and secrete correctly in B.subtilis.

The method for culturing the biofilm by using the solid MSGG is adopted, and the viscoelasticity of the biofilm is measured by using a rheometer (Antopa MCR101) (figure 11). The measurement method and parameters were the same as in example 1 above. Compared with a Db biomembrane, the biomembrane fused and expressed with BslA-3sp-spytag has greatly improved elasticity and little loss of toughness, but has reduced elasticity and toughness compared with the expression of pure BslA.

The method for culturing biofilms using the above solid MSGG and measuring the shear viscosity strength of biofilms using an universal mechanical device (Instron) (fig. 12). The measurement method and parameter settings were the same as in example 1. Meanwhile, 2.5. mu.L of tyrosinase of 1mmol/L is additionally added to the DB/pHT01-bsLA-3sp-spytag biomembrane, tyrosine rich in the fusion protein BslA-3sp-spytag is converted into catechol (DOPA) with excellent viscosity, and the modified shear viscosity is tested. The results show that the fusion protein BslA-3 sp-spytag-containing biofilm which was not treated with tyrosinase had a higher viscosity than Db, but was less viscous than Db biofilm expressing BslA protein alone. However, the viscosity of the tyrosinase-treated sample was greatly increased to about 250 kPa. This indicates that the fusion protein BslA-3sp-spytag can make the bacillus subtilis biofilm have better and more obvious viscosity improvement after being modified by tyrosinase.

In conclusion, the surface hydrophobin BslA of the bacillus subtilis is engineered, so that the adhesive capacity of the biological membrane can be enhanced, and the elasticity and toughness of the biological membrane can be improved to a certain extent.

Example 4: construction of integrated adhesive based on bacillus subtilis

On the basis of fully understanding the functions and characteristics of the components of the bacillus subtilis biomembrane, different viscosity characteristics such as amyloid protein, mussel byssus protein, positive and negative charge interaction substances and the like are modularly designed in the bacillus subtilis, so that the viscosity of the generated biomembrane can be improved. Therefore, by further integrating all the viscosity characteristics into the genome, the final T5B3 strain was obtained according to the above strain construction method, and further, in order to maximize the advantage of mussel byssus protein, by constructing tyrosinase for heterologous expression in bacillus subtilis (fig. 13), the T5B3/ph 01-tyrosinase strain (T5B 3 strain carrying the ph T01-tyrosinase plasmid) was obtained. And finally, identifying the adhesive capacity of the integrated biological membrane adhesive by adopting a rheological test and a universal mechanical device tensile test.

The method for culturing the biofilm by using the solid MSGG is adopted, and the viscoelasticity of the biofilm is measured by using a rheometer (Antopa MCR101) (FIG. 14). The measurement method and parameters were the same as in example 1. It can be seen that the storage modulus of the integrative strain T5B3/pHT01-Tyrosinase is all greater than that of the biofilm obtained from the previous modular design, indicating that the integration of all the adhesive characteristics facilitates the construction of a more powerful adhesive. However, expression of tyrosinase can reduce the toughness of the biofilm to some extent, thereby reducing adhesive recoverability.

The method for culturing biofilms using the above solid MSGG and measuring the shear viscometric strength of the biofilm using an universal mechanical apparatus (Instron) (fig. 15). The measurement method and parameter settings were the same as in example 1. It can be seen that the viscosity of the integrated T5B3/pHT01-tyrosinase biofilm is increased by adding Fe³⁺The adhesive force is greatly improved to 255.76 +/-39.24 kPa, and is higher than that of the biological film with partial adhesive characteristic of the previous modular designThe sexual performance is high.

In summary, by further integration of a single modular design, a final more viscous biofilm strain can be obtained. In addition, compared with the modular design of a plasmid series, the final genome integration reduces the risk of plasmid loss, improves the stability of the strain, and has more practical application advantages.

Example 5: performance and application of integrated adhesive based on bacillus subtilis

For the construction of the complete integrated Bacillus subtilis T5B3/pHT01-tyrosinase, the performance and the practicability of the integrated Bacillus subtilis T5B3/pHT01-tyrosinase are shown by taking several examples, and in the examples, the materials are all biological membranes cultured by the solid MSGG.

As shown in FIG. 16a, the change in viscosity of the integrated biofilm adhesive after treatment with a detergent (sodium dodecyl sulfate (SDS), Urea (Urea)) was measured using a wild-type Bacillus subtilis biofilm (WT) as a control. As can be seen, both detergents have some effect on the viscosity of the biofilm. Overall, the control group showed a maximum reduction in viscosity of 55.1%, whereas the integrated biofilm plus Fe3+ showed a much reduced viscosity strength of only 20.9%. Whereas the wild-type biofilm showed a 81.7% reduction in tack at up to 90% relative humidity, the integrated adhesive material was only 40.6% (fig. 16 b). It can be seen that the integration adhesive has better environmental tolerance, probably due to the detergent only denaturing the protein and not destroying the PO in the EPS exopolysaccharide₃ ^2-The group can still exert the adhesive effect by the electrostatic interaction mediated by EPS-Fe3+, and in addition, the DOPA cross-linked structure formed in the biological membrane and Fe³⁺The dehydration and coagulation effects on the biofilm can make the structure of the biofilm denser, so that the biofilm can resist the damage of high-humidity environment to viscosity.

The shear viscosity of the biofilm between different passages is compared by re-streaking the solid cultured biofilm on LB solid medium and culturing a new biofilm on the basis of the re-streaking, and thus the biofilm of 5 generations grows repeatedly. It can be seen that when Fe is not added³⁺In time, the biofilm is 1-4 generations later (1)^st-2^nd-3^rd-4^th) The shear viscosity of (a) is substantially constant at about 150 kPa; and with Fe³⁺The viscosity after the action was maintained substantially between 200 and 260kPa (FIG. 17), although it fluctuated. And fifth generation (5)^th) Cultured biofilm supplemented with Fe³⁺The shear viscosity after the action then dropped to 189.24. + -. 22.61 kPa. It is speculated that the possibility of greater variation in biofilm composition during the subculture growth process is EPS, which on the one hand is beneficial for maintaining the toughness of the biofilm and on the other hand can also react with Fe³⁺Electrostatic interactions occur resulting in a significant increase in viscosity. In general, the biofilm adhesive material has better renewability, which reduces the cost for the use of the material.

As shown in fig. 18a, the biofilm can be extruded in a shape, such as the letter "b. It can be seen that biofilm is a very soft, highly plastic gel material, which provides convenience for storage and use of the material, especially when it is necessary to fill or bond places such as cracks, pores, etc. that are difficult to directly contact with using an adhesive.

Fig. 19a is a schematic diagram of filling cracks. By applying the constructed adhesive to various structures where cracks occur, the biofilm can adhere to the cracks as a soft material by virtue of its strong toughness and viscosity, and maintain good viscosity under water. As shown in FIG. 19b, the appropriate amount of biofilm was scraped off the MSGG solid plate, which was coated on one end of the slide glass, one end of a thin wire was fixed, the coated area was about 1X 1cm, the length of the fixed thin wire was about 1cm, and then 1mM FeCl in excess was added dropwise₃The solution is fully reacted with the biomembrane adhesive, and the excessive FeCl is absorbed by absorbent paper₃And (3) solution. The other end of the thread was lifted and the slide was placed in a 500mL beaker containing 500mL of water, and it was seen that the slide was still able to remain adhered to the thread in the water. In addition, as shown in FIG. 19c, a proper amount of cultured T5B3/pHT01-tyrosinase Bacillus subtilis biofilm was scraped off, evenly applied to the fractured surface of two fractured bones, and the fractured surface coated with the biofilm adhesive was pressedAfter 2min, the bone was held at one end and the fracture immersed in a 500mL beaker containing 500mL of water, it was seen that the two fractured bones remained tightly connected together and did not break.

In summary, the integrated underwater adhesive developed by the patent has better environmental tolerance, reproducibility, injectability and stronger underwater adhesion to different substrates, thereby being beneficial to using the biomembrane living body adhesive material with low cost, high efficiency and convenience in different environments.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

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tcagagttaa atggtattgc ttcactgctt catcttttct tttacggtcc catacttttt 60

gtttgaacag tacctgtgcg agcgggtacc ttttttttgc ttcttttaca gcaatctctt 120

cccatttgga catgtggcgg gcggttacaa gcggtgtttc ttctgcgtga gcggctgtgg 180

tgccaaagac gagaagagat agacaaatca cacattgttt gatcatcatg ctgtcaccct 240

ttctttgttt attattacca aataataatg ggatatgcat ttaaattctc acataacaat 300

cccaaaaatt tctaaaaaat tgaaaaaatg agcaatactg agcaagactt tgtaatatga 360

tgaaaacatt cttttaaacg aacaaaatga gcgatttcgg tgtttttaaa tctataaatc 420

gttgattata ctctatttgt gaagttcttt aaagagaacg attgtcatat caagttacag 480

tgttttacag gaggtaagat atgtttcgat tgtttcacaa tcagcaaaag gcgaagacga 540

aactgaaagt tctgcttatc tttcagcttt cagtcatttt cagtctgact gccgcaatat 600

gcttacaatt ttccgatgat acaagcgctg cttttcatga tattgaaaca tttgatgtct 660

cacttcaaac gtgtaaagac tttcagcata cagataaaaa ctgccattat gataaacgct 720

gggatcaaag tgatttgcac atatcagatc aaacggatac gaaaggcact gtatgctcac 780

ctttcgcctt atttgctgtg ctcgaaaata caggtgagaa acttaagaaa tcaaagtgga 840

agtgggagct tcataagctt gaaaatgccc gcaaaccgtt aaaggatggg aacgtgatcg 900

aaaaaggatt tgtctccaat caaatcggcg attcacttta taaaattgag accaagaaaa 960

aaatgaaacc cggcatttat gcatttaaag tatataaacc ggcaggctac ccggcaaacg 1020

gcagtacatt tgagtggtcg gagcctatga ggcttgcaaa atgcgatgaa aaaccgacag 1080

tccctaaaaa agaaacaaag tcggacgtca aaaaggagaa tgaaacaaca caaaaagata 1140

taccggaaaa aacaatgaaa gaagaaacat ctcaagaagc tgtaaccaaa gaaaaagaaa 1200

ctcaatcaga ccagaaggaa agcggggaag aggatgaaaa aagcaatgaa gctgatcagt 1260

aatattttat acgtgatcat ctttactctt attattgtgc tgacacttgt cgtgatttca 1320

acacgttcat ccgggggaga gccggcagtg tttgggtata cgctgaaatc agttctgtca 1380

ggttcgatgg agccggagtt caatacaggt tccttaatat tggtcaaaga aatcactgat 1440

gtgaaagagc tccaaaaagg tgacgttatt acatttatgc aggatgcaaa tacggcggtc 1500

acccacagaa ttgttgacat aacaaagcaa ggagaccatt tgttatttaa aacaaaaggt 1560

gataataatg cagcagctga ttcagcgcct gtatcggacg aaaatgttcg cgcgcaatac 1620

acaggttttc agcttccata tgccggctat atgcttcatt ttgccagcca gccgattgga 1680

acggctgtat tattgattgt tcccggcgtg atgctgttag tttacgcttt tgtgacgatc 1740

agcagcgcca ttagagaaat tgaaagaaag acaaaagcct tggaaacaga tacaaaggac 1800

agcaccatgt ctacttaact tcagttgtaa acctggcaac aggtttcgat ataaaatcat 1860

tcaataaaag gggagcttac catgggtatg aaaaagaaat tgagtttagg agttgcttct 1920

gcagcactag gattagcttt agttggagga ggaacatggg cagcatttaa cgacattaaa 1980

tcaaaggatg ctacttttgc atcaggtacg cttgatttat ctgctaaaga gaattcagcg 2040

agtgtgaact tatcaaatct aaagccggga gataagttga caaaggattt ccaatttgaa 2100

aataacggat cacttgcgat caaagaagtt ctaatggcgc ttaattatgg agattttaaa 2160

gcaaacggcg gcagcaatac atctccagaa gatttcctca gccagtttga agtgacattg 2220

ttgacagttg gaaaagaggg cggcaatggc tacccgaaaa acattatttt agatgatgcg 2280

aaccttaaag acttgtattt gatgtctgct aaaaatgatg cagcggctgc tgaaaaaatc 2340

aaaaaacaaa ttgaccctaa attcttaaat gcaagcggta aagtcaatgt agcaacaatt 2400

gatggtaaaa ccgctcctga atatgatggt gttccaaaaa caccaactga cttcgatcag 2460

gttcaaatgg aaatccaatt caaggatgat aaaacaaaag atgaaaaagg gcttatggtt 2520

caaaataaat atcaaggcaa ctccattaag cttcaattct cattcgaagc tacacagtgg 2580

aacggcttga caatcaaaaa ggaccatact gataaagatg gttacgtgaa agaaaatgaa 2640

aaagcgcata gcgaggataa aaattaataa 2670

<210> 14

<211> 2943

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tcagagttaa atggtattgc ttcactgctt catcttttct tttacggtcc catacttttt 60

gtttgaacag tacctgtgcg agcgggtacc ttttttttgc ttcttttaca gcaatctctt 120

cccatttgga catgtggcgg gcggttacaa gcggtgtttc ttctgcgtga gcggctgtgg 180

tgccaaagac gagaagagat agacaaatca cacattgttt gatcatcatg ctgtcaccct 240

ttctttgttt attattacca aataataatg ggatatgcat ttaaattctc acataacaat 300

cccaaaaatt tctaaaaaat tgaaaaaatg agcaatactg agcaagactt tgtaatatga 360

tgaaaacatt cttttaaacg aacaaaatga gcgatttcgg tgtttttaaa tctataaatc 420

gttgattata ctctatttgt gaagttcttt aaagagaacg attgtcatat caagttacag 480

tgttttacag gaggtaagat atgtttcgat tgtttcacaa tcagcaaaag gcgaagacga 540

aactgaaagt tctgcttatc tttcagcttt cagtcatttt cagtctgact gccgcaatat 600

gcttacaatt ttccgatgat acaagcgctg cttttcatga tattgaaaca tttgatgtct 660

cacttcaaac gtgtaaagac tttcagcata cagataaaaa ctgccattat gataaacgct 720

gggatcaaag tgatttgcac atatcagatc aaacggatac gaaaggcact gtatgctcac 780

ctttcgcctt atttgctgtg ctcgaaaata caggtgagaa acttaagaaa tcaaagtgga 840

agtgggagct tcataagctt gaaaatgccc gcaaaccgtt aaaggatggg aacgtgatcg 900

aaaaaggatt tgtctccaat caaatcggcg attcacttta taaaattgag accaagaaaa 960

aaatgaaacc cggcatttat gcatttaaag tatataaacc ggcaggctac ccggcaaacg 1020

gcagtacatt tgagtggtcg gagcctatga ggcttgcaaa atgcgatgaa aaaccgacag 1080

tccctaaaaa agaaacaaag tcggacgtca aaaaggagaa tgaaacaaca caaaaagata 1140

taccggaaaa aacaatgaaa gaagaaacat ctcaagaagc tgtaaccaaa gaaaaagaaa 1200

ctcaatcaga ccagaaggaa agcggggaag aggatgaaaa aagcaatgaa gctgatcagt 1260

aatattttat acgtgatcat ctttactctt attattgtgc tgacacttgt cgtgatttca 1320

acacgttcat ccgggggaga gccggcagtg tttgggtata cgctgaaatc agttctgtca 1380

ggttcgatgg agccggagtt caatacaggt tccttaatat tggtcaaaga aatcactgat 1440

gtgaaagagc tccaaaaagg tgacgttatt acatttatgc aggatgcaaa tacggcggtc 1500

acccacagaa ttgttgacat aacaaagcaa ggagaccatt tgttatttaa aacaaaaggt 1560

gataataatg cagcagctga ttcagcgcct gtatcggacg aaaatgttcg cgcgcaatac 1620

acaggttttc agcttccata tgccggctat atgcttcatt ttgccagcca gccgattgga 1680

acggctgtat tattgattgt tcccggcgtg atgctgttag tttacgcttt tgtgacgatc 1740

agcagcgcca ttagagaaat tgaaagaaag acaaaagcct tggaaacaga tacaaaggac 1800

agcaccatgt ctacttaact tcagttgtaa acctggcaac aggtttcgat ataaaatcat 1860

tcaataaaag gggagcttac catgggtatg aaaaagaaat tgagtttagg agttgcttct 1920

gcagcactag gattagcttt agttggagga ggaacatggg cagcatttaa cgacattaaa 1980

tcaaaggatg ctacttttgc atcaggtacg cttgatttat ctgctaaaga gaattcagcg 2040

agtgtgaact tatcaaatct aaagccggga gataagttga caaaggattt ccaatttgaa 2100

aataacggat cacttgcgat caaagaagtt ctaatggcgc ttaattatgg agattttaaa 2160

gcaaacggcg gcagcaatac atctccagaa gatttcctca gccagtttga agtgacattg 2220

ttgacagttg gaaaagaggg cggcaatggc tacccgaaaa acattatttt agatgatgcg 2280

aaccttaaag acttgtattt gatgtctgct aaaaatgatg cagcggctgc tgaaaaaatc 2340

aaaaaacaaa ttgaccctaa attcttaaat gcaagcggta aagtcaatgt agcaacaatt 2400

gatggtaaaa ccgctcctga atatgatggt gttccaaaaa caccaactga cttcgatcag 2460

gttcaaatgg aaatccaatt caaggatgat aaaacaaaag atgaaaaagg gcttatggtt 2520

caaaataaat atcaaggcaa ctccattaag cttcaattct cattcgaagc tacacagtgg 2580

aacggcttga caatcaaaaa ggaccatact gataaagatg gttacgtgaa agaaaatgaa 2640

aaagcgcata gcgaggataa aaatggagga ggaggctcag gatccagcag cgaagagtat 2700

aagggcggct attatcctgg taatgcgtat cactaccatt caggcggcag ctatcatggc 2760

tcaggctatc atggcggcta taagggcaaa tactacggca aggcgaaaaa atattattat 2820

aaatataaaa atagcggcaa gtataaatat ctgaaaaaag cgcgcaaata ccatcgcaag 2880

ggctataagt attacggcgg cagcagcact agtcaccatc atcaccatca tcattggcca 2940

taa 2943

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acaattccca attaaaggag g 21

<210> 16

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ctgccccggg gacgtcgact ctagattatt aatttttatc ctcgctatgc 50

<210> 17

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacgtcgact ctagaggatc cttatggcca atgatgatgg tga 43

<210> 18

<211> 546

<212> DNA

<213> Bacillus subtilis

<400> 18

atgaaacgca aattattatc ttctttggca attagtgcat taagtctcgg gttactcgtt 60

tctgcaccta cagcttcttt cgcggctgaa tctacatcaa ctaaagctca tactgaatcc 120

actatgagaa cacagtctac agcttcattg ttcgcaacaa tcactggcgc cagcaaaacg 180

gaatggtctt tctcagatat cgaattgact taccgtccaa acacgcttct cagccttggc 240

gttatggagt ttacattgcc aagcggattt actgcaaaca cgaaagacac attgaacgga 300

aatgccttgc gtacaacaca gatcctcaat aacgggaaaa cagtaagagt tcctttggca 360

cttgatttgt taggagctgg cgaattcaaa ttaaaactga ataacaaaac acttcctgcc 420

gctggtacat atactttccg tgcggagaat aaatcattaa gcatcggaaa taaattttac 480

gcagaagcca gcattgacgt ggctaagcgc agcactcctc cgactcagcc ttgcggttgc 540

aactaa 546

<210> 19

<211> 675

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgaaacgca aattattatc ttctttggca attagtgcat taagtctcgg gttactcgtt 60

tctgcaccta cagcttcttt cgcggctgaa tctacatcaa ctaaagctca tactgaatcc 120

actatgagaa cacagtctac agcttcattg ttcgcaacaa tcactggcgc cagcaaaacg 180

gaatggtctt tctcagatat cgaattgact taccgtccaa acacgcttct cagccttggc 240

gttatggagt ttacattgcc aagcggattt actgcaaaca cgaaagacac attgaacgga 300

aatgccttgc gtacaacaca gatcctcaat aacgggaaaa cagtaagagt tcctttggca 360

cttgatttgt taggagctgg cgaattcaaa ttaaaactga ataacaaaac acttcctgcc 420

gctggtacat atactttccg tgcggagaat aaatcattaa gcatcggaaa taaattttac 480

gcagaagcca gcattgacgt ggctaagcgc agcactcctc cgactcagcc ttgcggttgc 540

aacggaagcg gctacgatgg ctataactgg ccgtatggct acaacggcta ccgctatggc 600

tggaacaaag gctggaatgg ctatggaagc gtcccgacaa tcgtcatggt cgacgcctac 660

aaacgctaca aataa 675

<210> 20

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tcccattaaa ggaggaagga tccatgaaac gcaaattatt a 41

<210> 21

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ccgctcatta ggcgggctgc cccgggttaa tgatggtgat ggtgatggtt gcaaccgcaa 60

ggctgagtc 69

<210> 22

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ttaggcgggc tgccccgggt tatttgtagc gtttgtaggc gtcga 45

<210> 23

<211> 62

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ttacacatta actagacaga tctatcgatg catgccatgg aaaccagaaa gcggacttaa 60

gc 62

<210> 24

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

taacagcaaa aaaaagagac ggccc 25

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atgggtatga aaaagaaatt g 21

<210> 26

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tatgaatact gggccgtctc ttttttttgc tgttattatg gccaatgatg atggtg 56

<210> 27

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

agcaactcct aaactcaatt tc 22

<210> 28

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tctgcagaag cttctagaat tcgagctccc gggtaaaaca aaaggtgata ataa 54

<210> 29

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

acagatctat cgatgcatgc catggtgcgt gaggagttgc aaatgagcaa 50

<210> 30

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gataataatt tgcgtttcat aacaaaattc cccctaaaaa 40

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

atgaaacgca aattattatc 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ttaatagcca ttccagcctt 20

<210> 33

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

tggaacaaag gctggaatgg ctattaatgt aaaagaccgg ttaacgccgg tct 53

<210> 34

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

agcttctaga attcgagctc ccgggatctc catgatgttt aatggccagg 50

<210> 35

<211> 1170

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccaa actgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggcg 720

ggtggcggtg gtggtagcgg tggtggcggt agctcgtact accatcacca tcaccatcac 780

gattacgaca tcccaacgac cgaaaacctg tattttcagg gcgccatggt aaccacctta 840

tcaggtttat caggtgagca aggtccgtcc ggtgatatga caactgaaga agatagtgct 900

acccatatta aattctcaaa acgtgatgag gacggccgtg agttagctgg tgcaactatg 960

gagttgcgtg attcatctgg taaaactatt agtacatgga tttcagatgg acatgtgaag 1020

gatttctacc tgtatccagg aaaatataca tttgtcgaaa ccgcagcacc agacggttat 1080

gaggtagcaa ctgctattac ctttacagtt aatgagcaag gtcaggttac tgtaaatggc 1140

gaagcaacta aaggtgacgc tcatacttaa 1170

<210> 36

<211> 840

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

atgacagtcc gtaaaaatca agcatccttg acggcggaag agaaacgtcg ttttgtagcg 60

gcattacttg aattaaaacg taccggccgt tatgacgcgt tcgtgactac ccacaatgca 120

ttcattctgg gggataccga taacggtgaa cgcacaggcc accgctcgcc cagctttctg 180

ccctggcatc gccgcttctt gttggagttt gaacgcgcac tgcaatcggt cgatgcgagc 240

gtcgccctgc cgtattggga ttggtccgcg gatcgctcaa cccgctcttc gctgtgggcg 300

ccagattttt tgggcggcac gggccgtagc cgcgacggcc aggttatgga tggccctttc 360

gcagcctcgg caggtaattg gccgattaat gtccgtgtgg acggccgcac gttcctccgc 420

cgtgcgttag gcgccggcgt gagcgaactg ccaacccgcg ctgaagtgga cagcgtcctg 480

gccatggcta cctacgatat ggcgccctgg aactccggca gcgatggctt tcgcaatcat 540

ttagagggtt ggcgcggtgt caatctgcat aaccgcgtgc acgtttgggt tggtggtcaa 600

atggccacgg gcgttagccc caatgatccg gttttctggt tacaccatgc ctatattgat 660

aaattatggg cggaatggca gcgccgtcat ccgagcagcc cgtatttgcc aggcggaggt 720

acgcccaacg tggtggactt aaatgaaact atgaagccgt ggaatgacac gacccccgca 780

gccctgttag atcatacccg tcactatacc tttgatgtcc atcaccatca ccaccattaa 840

<210> 37

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ttcccaatta aaggaggaag gatccatgac agtccgtaaa aatca 45

<210> 38

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

tgaaaaaagc ccgctcatta ggcgggctgc cccgggttaa tggtggtgat ggtgatgga 59

<210> 39

<211> 261

<212> PRT

<213> Bacillus subtilis

<400> 39

Met Gly Met Lys Lys Lys Leu Ser Leu Gly Val Ala Ser Ala Ala Leu

1 5 10 15

Gly Leu Ala Leu Val Gly Gly Gly Thr Trp Ala Ala Phe Asn Asp Ile

20 25 30

Lys Ser Lys Asp Ala Thr Phe Ala Ser Gly Thr Leu Asp Leu Ser Ala

35 40 45

Lys Glu Asn Ser Ala Ser Val Asn Leu Ser Asn Leu Lys Pro Gly Asp

50 55 60

Lys Leu Thr Lys Asp Phe Gln Phe Glu Asn Asn Gly Ser Leu Ala Ile

65 70 75 80

Lys Glu Val Leu Met Ala Leu Asn Tyr Gly Asp Phe Lys Ala Asn Gly

85 90 95

Gly Ser Asn Thr Ser Pro Glu Asp Phe Leu Ser Gln Phe Glu Val Thr

100 105 110

Leu Leu Thr Val Gly Lys Glu Gly Gly Asn Gly Tyr Pro Lys Asn Ile

115 120 125

Ile Leu Asp Asp Ala Asn Leu Lys Asp Leu Tyr Leu Met Ser Ala Lys

130 135 140

Asn Asp Ala Ala Ala Ala Glu Lys Ile Lys Lys Gln Ile Asp Pro Lys

145 150 155 160

Phe Leu Asn Ala Ser Gly Lys Val Asn Val Ala Thr Ile Asp Gly Lys

165 170 175

Thr Ala Pro Glu Tyr Asp Gly Val Pro Lys Thr Pro Thr Asp Phe Asp

180 185 190

Gln Val Gln Met Glu Ile Gln Phe Lys Asp Asp Lys Thr Lys Asp Glu

195 200 205

Lys Gly Leu Met Val Gln Asn Lys Tyr Gln Gly Asn Ser Ile Lys Leu

210 215 220

Gln Phe Ser Phe Glu Ala Thr Gln Trp Asn Gly Leu Thr Ile Lys Lys

225 230 235 240

Asp His Thr Asp Lys Asp Gly Tyr Val Lys Glu Asn Glu Lys Ala His

245 250 255

Ser Glu Asp Lys Asn

260

<210> 40

<211> 181

<212> PRT

<213> Bacillus subtilis

<400> 40

Met Lys Arg Lys Leu Leu Ser Ser Leu Ala Ile Ser Ala Leu Ser Leu

1 5 10 15

Gly Leu Leu Val Ser Ala Pro Thr Ala Ser Phe Ala Ala Glu Ser Thr

20 25 30

Ser Thr Lys Ala His Thr Glu Ser Thr Met Arg Thr Gln Ser Thr Ala

35 40 45

Ser Leu Phe Ala Thr Ile Thr Gly Ala Ser Lys Thr Glu Trp Ser Phe

50 55 60

Ser Asp Ile Glu Leu Thr Tyr Arg Pro Asn Thr Leu Leu Ser Leu Gly

65 70 75 80

Val Met Glu Phe Thr Leu Pro Ser Gly Phe Thr Ala Asn Thr Lys Asp

85 90 95

Thr Leu Asn Gly Asn Ala Leu Arg Thr Thr Gln Ile Leu Asn Asn Gly

100 105 110

Lys Thr Val Arg Val Pro Leu Ala Leu Asp Leu Leu Gly Ala Gly Glu

115 120 125

Phe Lys Leu Lys Leu Asn Asn Lys Thr Leu Pro Ala Ala Gly Thr Tyr

130 135 140

Thr Phe Arg Ala Glu Asn Lys Ser Leu Ser Ile Gly Asn Lys Phe Tyr

145 150 155 160

Ala Glu Ala Ser Ile Asp Val Ala Lys Arg Ser Thr Pro Pro Thr Gln

165 170 175

Pro Cys Gly Cys Asn

180

<210> 41

<211> 10

<212> PRT

<213> Mytilus edulis

<400> 41

Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys

1 5 10

<210> 42

<211> 42

<212> PRT

<213> Mytilus galloprovincialis

<400> 42

Thr Asp Lys Ala Tyr Lys Pro Asn Pro Cys Val Val Ser Lys Pro Cys

1 5 10 15

Lys Asn Arg Gly Lys Cys Ile Trp Asn Gly Lys Ala Tyr Arg Cys Lys

20 25 30

Cys Ala Tyr Gly Tyr Gly Gly Arg His Cys

35 40

<210> 43

<211> 48

<212> PRT

<213> Mytilus edulis

<400> 43

Ala Asp Tyr Tyr Gly Pro Asn Tyr Gly Pro Pro Arg Arg Tyr Gly Gly

1 5 10 15

Gly Asn Tyr Asn Arg Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr

20 25 30

Lys Gly Trp Asn Asn Gly Trp Asn Arg Gly Arg Arg Gly Lys Tyr Trp

35 40 45

<210> 44

<211> 74

<212> PRT

<213> Mytilus edulis

<400> 44

Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ala Tyr His

1 5 10 15

Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr

20 25 30

Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys

35 40 45

Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg

50 55 60

Lys Gly Tyr Lys Tyr Tyr Gly Gly Ser Ser

65 70

<210> 45

<211> 99

<212> PRT

<213> Mytilus californianus

<400> 45

Gly Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser

1 5 10 15

Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser

20 25 30

Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Cys Leu Pro Arg Asn Pro

35 40 45

Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys Pro Asp

50 55 60

Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn Asp Lys

65 70 75 80

Asp Tyr Gly Cys Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Arg

85 90 95

Ser Gly Tyr

<210> 46

<211> 47

<212> PRT

<213> Perna viridis

<400> 46

Gln Leu Thr Cys Phe Pro Thr Ile Asp Cys Gly Phe Asn Ile Asp Gly

1 5 10 15

Cys Gln Ser Phe Cys Arg Asp Arg Asn Cys Ser Pro Tyr Gly Ser Glu

20 25 30

Cys Arg Asn Asn Asn Leu Cys Cys Cys Leu Tyr Cys Arg Phe Gly

35 40 45

<210> 47

<211> 82

<212> PRT

<213> Perna viridis

<400> 47

Val Tyr Tyr Pro Asn Pro Cys Ser Pro Tyr Pro Cys Arg Asn Gly Gly

1 5 10 15

Thr Cys Lys Lys Arg Gly Leu Tyr Ser Tyr Lys Cys Tyr Cys Arg Lys

20 25 30

Gly Tyr Thr Gly Lys Asn Cys Gln Tyr Asn Ala Cys Phe Pro Asn Pro

35 40 45

Cys Leu Asn Gly Gly Thr Cys Gly Tyr Val Tyr Gly Tyr Pro Tyr Tyr

50 55 60

Lys Cys Ser Cys Pro Tyr Gly Tyr Tyr Gly Lys Gln Cys Gln Leu Lys

65 70 75 80

Lys Tyr

<210> 48

<211> 25

<212> PRT

<213> Mytilus californianus

<400> 48

Gly Tyr Asp Gly Tyr Asn Trp Pro Tyr Gly Tyr Asn Gly Tyr Arg Tyr

1 5 10 15

Gly Trp Asn Lys Gly Trp Asn Gly Tyr

20 25

<210> 49

<211> 41

<212> PRT

<213> Mytilus californianus

<400> 49

Gly Tyr Asp Gly Tyr Asn Trp Pro Tyr Gly Tyr Asn Gly Tyr Arg Tyr

1 5 10 15

Gly Trp Asn Lys Gly Trp Asn Gly Tyr Gly Ser Val Pro Thr Ile Val

20 25 30

Met Val Asp Ala Tyr Lys Arg Tyr Lys

35 40

<210> 50

<211> 45

<212> PRT

<213> Mytilus californianus

<400> 50

Gly Tyr Gly Tyr Asp Leu Gly Tyr Asn Ala Pro Trp Pro Tyr Asn Asn

1 5 10 15

Gly Tyr Tyr Gly Tyr Asn Gly Tyr Asn Gly Tyr His Gly Arg Tyr Gly

20 25 30

Trp Asn Lys Gly Trp Asn Asn Gly Pro Trp Gly Gly Tyr

35 40 45

Claims (11)

1. A composition comprising a TasA fusion protein comprising a TasA protein fragment and a mussel byssus protein fragment, an exopolysaccharide which is a metal cation chelated exopolysaccharide, and a BslA fusion protein comprising a BslA protein fragment and a self-aggregating adhesive protein fragment.

2. The composition of claim 1, further comprising one or more of the following technical features:

A1) the TasA protein fragment is: a) polypeptide fragment with amino acid sequence shown as SEQ ID No. 39; or b) a polypeptide fragment having an amino acid sequence with 80% or more homology to SEQ ID NO.39 and having the function of the polypeptide fragment defined in a);

A2) the mussel byssus protein is derived from mussels, and is selected from Mytilus edulis byssus protein, perna viridis byssus protein, Mytilus californica byssus protein and Mediterranean mussel byssus protein;

A3) the mussel byssus protein is selected from mefp-1, mgfp-2, mefp-3, mefp-5, mcfp-6, pvfp3 and pvfp 5; A4) the mussel byssus protein fragment is as follows: c) a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID Nos. 41 to 47, or d) a polypeptide fragment having an amino acid sequence with more than 80% homology with one of SEQ ID Nos. 41 to 47 and having the function of the polypeptide fragment defined in c);

A5) the TasA protein is derived from bacillus subtilis;

A6) the TasA fusion protein sequentially comprises a TasA protein fragment and a mussel byssus protein fragment from the N end to the C end;

A7) in the composition, the mass ratio of the TasA fusion protein to the extracellular polysaccharide to the BslA fusion protein is 1-5: 80-90: 1-5.

3. The composition of claim 1, further comprising one or more of the following technical features:

B1) the exopolysaccharide is a polyanionic polymer;

B2) the metal cation is selected from one or more of iron ion, magnesium ion, calcium ion and manganese ion;

B3) the exopolysaccharide is derived from bacillus.

4. The composition of claim 1, further comprising one or more of the following technical features:

C1) the BslA protein fragment is: e) polypeptide fragment with amino acid sequence shown as SEQ ID No. 40; or f) a polypeptide fragment having an amino acid sequence with 80% or more homology to SEQ ID NO.40 and having the function of the polypeptide defined in e);

C2) the self-agglomerating adhesive protein is selected from mussel byssus protein, california and derived adhesive short peptides;

C3) the self-agglomerating binding protein is selected from the group consisting of mcfp-3s, mcfp3 spep-spytag;

C4) the self-agglomerating adhesive protein fragments are: g) a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID Nos. 48 to 50, or h) a polypeptide fragment having an amino acid sequence with more than 80% homology to one of SEQ ID Nos. 48 to 50 and having the function of the polypeptide fragment defined in g);

C5) the BslA protein is derived from bacillus subtilis;

C6) the self-agglomerating adhesive protein is derived from mussel;

C7) the BslA fusion protein sequentially comprises a BslA fragment and a self-aggregating adhesive protein fragment from the N end to the C end;

C8) the BslA fusion protein is modified with tyrosinase.

5. A biofilm comprising a TasA fusion protein, exopolysaccharide, BslA fusion protein of a composition of any of claims 1-4.

6. The biofilm of claim 5, further comprising one or more of the following technical features:

D1) in the biological membrane, the content of the TasA fusion protein is 1-5 wt%;

D2) in the biological film, the content of extracellular polysaccharide is 80-90 wt%;

D3) in the biological membrane, the content of BslA fusion protein is 1-5 wt%;

D4) the biofilm is derived from bacillus.

7. An expression system capable of forming a biofilm according to any one of claims 5 to 6.

8. The expression system of claim 7, comprising an engineered bacterium, at least a portion of which comprises a construct capable of expressing the TasA fusion protein, or having integrated into its genome an exogenous polynucleotide encoding the TasA fusion protein;

at least part of the engineering bacteria contain a construction body capable of expressing the BslA fusion protein or a polynucleotide which is integrated with the genome and externally encodes the BslA fusion protein;

at least part of the engineering bacteria can produce EPS polysaccharide;

at least part of the engineering bacteria contains a construction body capable of expressing tyrosine or a polynucleotide which is integrated with exogenous tyrosine and codes for tyrosine in a genome;

the engineering bacteria are selected from bacillus.

9. A method of producing a biofilm according to any one of claims 5 to 6, comprising: culturing the expression system of any one of claims 7-8 under conditions suitable for the formation of said biofilm.

10. The expression system according to any one of claims 7 to 8 or the preparation process according to claim 9 expressing the obtained biofilm.

11. Use of a composition according to any of claims 1 to 4, a biofilm according to any of claims 5 to 6 in the preparation of an underwater adhesive.

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