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US 2024/0318215 A1Published application
Methods and compositions
OwnerKenneth Thomas Walker, Thomas Marc Ellisinventors
Life of this patent
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Abstract
The present invention relates to biomaterials, in particular bacterial cellulose and provides means to prepare pigmented cellulose at acidic pH 5.8, wherein cellulose pellicles comprising tyrosinase (EC 1.14.18.1) are melanated using a development solution at pH 6 to 8.5 and comprises tyrosine, cysteine and/or cystine. Further, the invention relates to means of preparing spatially restricted pigmented cellulose using an optogenetic expression system wherein two polymerase or transcription factor domains are split and each linked to a light-inducible dimerization domain. The invention provides corresponding methods and components.
Claims46
A method for producing melanated bacterial cellulose, wherein the method comprises exposing a cellulose pellicle that comprises tyrosinase to a development solution, wherein the development solution:
- is at a pH of between 6 and 8.5;
- comprises L-tyrosine and/or L-cysteine and/or L-cystine;
- and optionally comprises metal ions with an oxidation state of 2+;
- optionally wherein the cellulose pellicle was produced by bacterial cells that express tyrosinase.
1 more element
A method for producing melanated bacterial cellulose, wherein the method comprises:
- a) culturing a cellulose producing bacteria under conditions so as to allow a pellicle to form
- wherein the bacteria express tyrosinase;
- and
- b) exposing the pellicle formed in a) to a development solution;
- wherein the development solution:
- is at a pH of between 6 and 8.5;
- comprises L-tyrosine and/or L-cysteine and/or L-cystine;
- and optionally
- comprises metal ions with an oxidation state of 2+;
6 more elements
The method according to any of the preceding claims wherein the bacterial cells:
- a) are capable of producing bacterial cellulose;
- b) express all of bcsA, bcsD, bscC and bscD;
- c) belong to a genus selected from the group comprising or consisting of:
- Komagataeibacter, Escherichia, Gluconacetobacter, Acetobacter, Sarcina, Agrobacterium, Azotobacter, Rhizobium, Pseudomonas, Salmonella and Alcaligenes;
- d) are selected from the group comprising or consisting of:
- Komagaeibacter rhaeticus;
- Komagaeibacter xylinus, Komagaeibacter hansenii, Komagaeibacter medellinensis, Komagaelbacter europaeus, Komagaeibacter maltaceti, Komagaelbacter pomaceti, Komagaeibacter oboediens, or Komagaeibacter saccharivoans;
- e) are selected from the group comprising or consisting of:
- i) a strain of Komagaelbacter rhaeticus selected from the group comprising or consisting of:
- Komagaeibacter rhaeticus iGEM. Komagaeibacter rhaeticus AF;
- Komagaeibacter rhaeticus LMG22126;
- or
- ii) Gluconacetobacter xylinus CGMCC 2995;
- and/or
- f) are Komagaeibacter rhaeticus IGEM cells.
12 more elements
The method according to any of the preceding claims wherein the metal ions with an oxidation state of 2+ are selected from:
- a) Cu2+, Zn2+, Be2+, Mg2+, Ca2+, Cr2+, Mn2+, Co2+ or Ni2+;
- b) Cu2+, Zn2+;
- or
- c) Cu2+.
1 more element
The method according to any of the preceding claims wherein the development solution comprises:
- a) a water-soluble copper (II) salt, optionally comprises CuSO4 or CuCl2, optionally comprises:
- i) at least 2 μM CuSO4, optionally at least 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM or at least 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, or at least 160 μM CuSO4;
- and/or
- between 2 μM CuSO4 and 20 μM CuSO4;
- and/or
- less than 20 μM, optionally less than 17.5 μM, 15 μM, 12.5 μM, 10 μM, 7.5 μM, 5 μM, 4 μM, 3 μM, or less than 2 μM;
- and/or
- between 20 μM and 160 μM CuSO4;
- and/or
- less than 160 μM, optionally less than 140 μM, 120 μM, 100 μM, 80 μM, 60 μM, 40 μM, or less than 20 μM CuSO4;
- and/or
- ii) at least 2 μM CuCl2, optionally at least 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM or at least 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, or at least 160 μM CuCl2;
- and/or
- between 2 μM CuCl2 and 20 μM CuCl2;
- and/or
- less than 20 μM, optionally less than 17.5 μM, 15 μM, 12.5 μM, 10 μM, 7.5 μM, 5 μM, 4 μM, 3 μM, or less than 2 μM;
- and/or
- between 20 μM and 160 μM CuCl2;
- and/or
- less than 160 μM, optionally less than 140 μM, 120 μM, 100 μM, 80 μM, 60 μM, 40 μM, or less than 20 μM CuCl2;
- and/or
- b) at least 0.1 g/L tyrosine, optionally at least 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.75 g/L, 1 g/L, 1.5 g/L, 1.75 g/L or at least 2 g/L;
- between 0.1 g/L and 2 g/L tyrosine;
- and/or
- less than 2 g/L tyrosine, or less than 1.75 g/L, 1.5 g/L, 1 g/L, 0.75 g/L 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, or less than 0.1 g/L tyrosine;
- and/or
- c) at least 10 g/L cysteine, optionally at least 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L, 120 g/L, 140 g/L, 160 g/L, 180 g/L, 200 g/L, 220 g/L, 240 g/L, 260 g/L, 280 g/L or at least 300 g/L;
- between 10 g/L and 300 g/L cysteine;
- and/or
- less than 300 g/L cysteine, or less than 280 g/L, 260 g/L, 240 g/L, 220 g/L 200 g/L, 180 g/L, 160 g/L, 140 g/L, 120 g/L, 100 g/L, 90 g/L, 80 g/L 70 g/L, 60 g/L, 50 g/L, 40 g/L, 30 g/L, 20 g/L or less than 10 g/L cysteine;
- and/or
- d) at least 0.1 g/L L-cystine, optionally at least 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.75 g/L, 1 g/L, 1.5 g/L, 1.75 g/L or at least 2 g/L;
- between 0.1 g/L and 2 g/L L-cystine;
- and/or
- less than 2 g/L L-cystine, or less than 1.75 g/L, 1.5 g/L, 1 g/L, 0.75 g/L 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, or less than 0.1 g/L L-cystine.
- optionally wherein the development solution is at a pH of:
- at least 6, optionally at least 6.25, 6.5, 6.75, 7, 7.25, 7.4, 7.5, 7.75, 8, 8.25 or at least 8.5;
- and/or
- between 6 and 8.5, optionally between 6.25 and 8.25, 6.5 and 8, 6.25 and 7.75, 6.5 and 7.5, 6.75 and 7.25;
- or
- 7.4.
38 more elements
The method according to any of the preceding claims wherein the development solution comprises:
- a) PBS at pH 7.4;
- b) 10 μM CuSO4 or 20 μM CuSO4;
- and
- c) 0.5 g/L L-tyrosine or 1 g/L L-tyrosine, and/or 1 g/L L-cysteine and/or 0.4 g/L L-cystine.
1 more element
The method according to any of the preceding claims wherein the method comprises a further step of:
- (c) sterilising the pellicle following incubation in the development solution, optionally wherein the sterilisation is selected from the group comprising or consisting of:
- i) autoclaving;
- ii) heating;
- and/or
- iii) desiccation, optionally with 70% ethanol.
2 more elements
The method according to any of the preceding claims wherein the tyrosinase:
- a) Is a bacterial tyrosinase, optionally:
- Tyr1 from Bacillus megaterium [SEQ ID NO:
- 13];
- mel from Streptomyces antibiotics [SEQ ID NO:
- 14];
- or
- mel from Rhizobium etli [SEQ ID NO:
- 15];
- optionally wherein the tyrosinase comprises or consists of a sequence with at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or 100% sequence identity to any of the above sequences;
- and/or
- b) is operably linked to Anderson promoter J23104 [SEQ ID NO:
- 16] and RBS B0034 [SEQ ID NO:
- 17];
- optionally is operably linked to Anderson promoter J23104 and RBS B0034 that comprises or consists of a sequence with at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or 100% sequence identity to any of the above sequences.
11 more elements
The method according to any of the preceding claims wherein the cells are cultured in a culture medium that:
- i) is Hestrin and Schramm (HS) medium;
- ii) is supplemented with glucose, optionally at 2% (w/v);
- and/or
- iii) is buffered to a pH of 5.8.
1 more element
A nucleic acid comprising a regulatory sequence and a sequence that encodes a tyrosinase enzyme wherein the regulatory sequence comprises Anderson promoter J23104 and RBS B0034, optionally wherein:
- a) the sequence that encodes a tyrosinase enzyme encodes a bacterial tyrosinase, optionally:
- Tyr1 from Bacillus megaterium [SEQ ID NO:
- 13];
- mel from Streptomyces antibiotics [SEQ ID NO:
- 14];
- or
- mel from Rhizobium etli [SEQ ID NO:
- 15];
- optionally wherein the tyrosinase comprises or consists of a sequence with at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or 100% sequence identity to any of the above sequences;
- and/or
- b) the nucleic acid is a circular nucleic acid, optionally is a circular nucleic acid selected from the group consisting of a plasmid, a bacterial artificial chromosome, a phagemid, a cosmid, a yeast artificial chromosome, a human artificial chromosome, a viral vector, optionally
- wherein the circular nucleic acid, optionally a plasmid, further comprises an origin of replication;
- and/or
- c) wherein any one or more of the nucleic acids further comprises a selectable marker;
- and/or
- d) the nucleic acid is integrated into the genome of a cell, optionally a bacterial cell.
13 more elements
An optogenetic expression system for use in bacteria of the genus Komagataeibacter, comprising:
- (a) A first nucleic acid comprising a first nucleotide sequence that encodes a first polypeptide, wherein the first polypeptide comprises:
- i) a first domain that comprises a first portion of a heterologous split-polymerase or a split-transcription factor;
- and
- ii) a second domain that comprises a first light-inducible dimerization domain;
- (b) A second nucleic acid comprising a second nucleotide sequence that encodes a second polypeptide, wherein the second polypeptide comprises:
- i) A first domain that comprises a second portion of a heterologous split-polymerase or a split-transcription factor;
- and
- ii) A second domain that comprises a second light-inducible dimerization domain;
- and
- (c) A third nucleic acid comprising a third nucleic acid sequence that encodes a target protein or RNA to be expressed operably linked to a target promoter;
- and wherein the first light-inducible dimerization domain and the second light-inducible dimerization domain are capable of dimerising with one another upon exposure to light of a dimerization wavelength to form a functional heterologous polymerase or a functional transcription factor capable of transcribing or initiating transcription from the target promoter,
- and wherein the target promoter is recognised by the functional heterologous polymerase or functional transcription factor so as to drive transcription of the third nucleic acid sequence that encodes a target protein or RNA.
9 more elements
A spatially pigmented bacterial pellicle as produced according to any method of any of the preceding claims, optionally wherein the pigment is melanin.
A pigmented bacterial pellicle as produced according to any method of any of the preceding claims, optionally wherein the pigment is melanin.
A pigment development solution, wherein the solution:
- a) is at a pH of:
- between 6 and 8.5, optionally between 6.25 and 8.25, 6.5 and 8, 6.25 and 7.75, 6.5 and 7.5, 6.75 and 7.25;
- and/or
- at least 6, optionally at least 6.25, 6.5, 6.75, 7, 7.25, 7.4, 7.5, 7.75, 8, 8.25 or at least 8.5;
- and/or
- 7.4;
- b) comprises L-tyrosine and/or L-cysteine and/or L-cystine;
- optionally comprises:
- i) at least 0.1 g/L tyrosine, optionally at least 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.75 g/L, 1 g/L, 1.5 g/L, 1.75 g/L or at least 2 g/L;
- between 0.1 g/L and 2 g/L tyrosine;
- and/or
- less than 2 g/L tyrosine, or less than 1.75 g/L, 1.5 g/L, 1 g/L, 0.75 g/L 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, or less than 0.1 g/L tyrosine;
- and/or
- ii) at least 10 g/L cysteine, optionally at least 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L, 120 g/L, 140 g/L, 160 g/L, 180 g/L, 200 g/L, 220 g/L, 240 g/L, 260 g/L, 280 g/L or at least 300 g/L;
- between 10 g/L and 300 g/L cysteine;
- and/or
- less than 300 g/L cysteine, or less than 280 g/L, 260 g/L, 240 g/L, 220 g/L 200 g/L, 180 g/L, 160 g/L, 140 g/L, 120 g/L, 100 g/L, 90 g/L, 80 g/L 70 g/L, 60 g/L, 50 g/L, 40 g/L, 30 g/L, 20 g/L or less than 10 g/L cysteine;
- and/or
- iii) at least 0.1 g/L L-cystine, optionally at least 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.75 g/L, 1 g/L, 1.5 g/L, 1.75 g/L or at least 2 g/L;
- between 0.1 g/L and 2 g/L L-cystine;
- and/or
- less than 2 g/L L-cystine, or less than 1.75 g/L, 1.5 g/L, 1 g/L, 0.75 g/L 0.5 g/L, 0.4 g/L, 0.3 g/L, 0.2 g/L, or less than 0.1 g/L L-cystine;
- and optionally
- c) comprises metal ions with an oxidation state of 2+;
- optionally comprises:
- i) a) Cu2+, Zn2+, Be2+, Mg2+, Ca2+, Cr2+, Mn2+, Co2+ or Ni2+;
- b) Cu2+, Zn2+;
- and/or
- c) Cu2+;
- and/or
- ii) CUSO4, optionally comprises:
- at least 2 μM CuSO4, optionally at least 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM or at least 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, or at least 160 μM CuSO4;
- and/or
- between 2 μM CUSO4 and 20 μM CUSO4;
- and/or
- less than 20 μM, optionally less than 17.5 μM, 15 μM, 12.5 μM, 10 μM, 7.5 μM, 5 μM, 4 μM, 3 μM, or less than 2 μM;
- and/or
- between 20 μM and 160 μM CuSO4;
- and/or
- less than 160 μM, optionally less than 140 μM, 120 μM, 100 μM, 80 μM, 60 μM, 40 μM, or less than 20 μM CuSO4;
- and/or
- iii) CuCl2, optionally comprises:
- at least 2 μM CUCl2, optionally at least 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM or at least 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, or at least 160 μM CuCl2;
- and/or
- between 2 μM CuCl2 and 20 μM CUCl2;
- and/or
- less than 20 μM, optionally less than 17.5 μM, 15 μM, 12.5 μM, 10 μM, 7.5 μM, 5 μM, 4 μM, 3 μM, or less than 2 μM;
- between 20 μM and 160 μM CuCl2;
- and/or
- less than 160 μM, optionally less than 140 μM, 120 μM, 100 μM, 80 μM, 60 μM, 40 μM, or less than 20 μM CuCl2;
- optionally wherein the development solution comprises:
- a) PBS at pH 7.4;
- b) 10 μM CuSO4 or 20 μM CuSO4;
- and
- c) 0.5 g/L L-tyrosine, 1 g/L L-tyrosine, and/or 1 g/L cysteine and/or 0.4 g/L cystine.
52 more elements
A kit comprising:
- a) a bacterial pellicle as described in any of the above claims;
- and
- b) a development solution according to any of the preceding claims;
- and optionally comprises
- c) a light source capable of emitting light of the wavelength of the light results in dimerization of the first and second dimerization domains,
- optionally wherein the bacterial pellicle comprises an optogenetic expression system according to any of the preceding claims.
3 more elements
Dependencies are inferred from the claim text.