MODIFIED BACTERIAL NANOCELLULOSE AND ITS USES IN CHIP CARDS AND MEDICINE

Abstract

The present invention relates to bacterial nanocellulose composite which comprises nanocellulose, sensor or signal processing molecule(s), actuator/effector molecule(s) and/or cells and optionally further component(s). The present invention further relates to the use of the bacterial nanocellulose composite in chip technology and material engineering. The present invention relates to a printing, storage and/or processing medium as well as a smart card or chip card comprising the bacterial nanocellulose composite. The present invention further relates to the medical use of the bacterial nanocellulose composite, preferably in wound healing, tissue engineering and as transplant. The present invention further relates to a skin, tissue or neuro transplant. The present invention also relates to methods of stimulus conduction, muscle stimulation and/or monitoring heartbeat. The present invention further relates to a method for producing a nanocellulose composite chip using 3D printer.

Claims

1. A bacterial nanocellulose composite, said bacterial nanocellulose composite comprising nanocellulose and (i) sensor or signal processing molecule(s); and/or (ii) actuator or effector molecule(s); and/or (iii) cells.

2. The bacterial nanocellulose composite of claim 1, wherein the bacterial nanocellulose is obtained via bacterial fermentation or bacterial expression of gram-negative bacteria, Komagataeibacter, cyanobacteria, or from plant sources but then bacterially fermented.

3. The bacterial nanocellulose composite of claim 1, comprising a light-inducible or light-responding sensor/actuator/effector molecule(s) or light-inducible or a light-responding sensor/actuator/effector domain(s) comprising: blue light using FAD domain (BLUF domain), light-oxygen voltage sensing domain (LOV domain), or cryptochromes (CRYs).

4. The bacterial nanocellulose composite of claim 1, wherein the sensor or signal processing molecule(s) (i) are protein(s) comprising light-inducible or light-responding sensor domains which are selected from: polymerase(s); adenyltransferase(s); ion channel(s) or pore(s); membrane protein(s), lipoprotein(s), glycoprotein(s); receptors; enzyme; or domains thereof; or combinations thereof.

5. The bacterial nanocellulose composite of claim 1, wherein the actuator or effector molecule(s) (ii) are proteins selected from polymerase(s); exonuclease(s); transcription factor(s); nucleotide binding domain(s); enzyme(s); structural protein(s); protein translation enzyme(s); or domains thereof, or combinations thereof.

6. The bacterial nanocellulose composite of claim 3, wherein the protein(s) comprising light-inducible or light-responding sensor domain(s) further comprise linker(s) and/or secretion signal(s) or signal peptide domain(s).

7. The bacterial nanocellulose composite of claim 1, wherein sensor or signal processing molecule(s) (i) and/or the actuator or effector molecule(s) (ii) comprise or are fused to fluorescent protein(s) or protein domain(s) comprising fluorescent domain(s).

8. The bacterial nanocellulose composite of claim 1, wherein the bacterial nanocellulose further comprises components for the sensor/actuator molecule(s) (i) further polymer(s), graphene or fullerene, compounds supporting wound healing and/or stimulating growth, markers or labels, drugs, antibodies or antibody fragments, or combinations thereof.

9. The bacterial nanocellulose composite of claim 8, wherein the sensor or signal processing molecule(s) (i) and/or actuator or effector molecule(s) (ii) and/or cell(s) (iii) and/or further component(s) (iv), if present, are embedded or encapsulated, or the sensor or signal processing molecule(s) (i) and/or actuator or effector molecule(s) (ii) and/or further component(s) (iv), if present, are covalently attached to the nanocellulose, such as via a linker, anchor groups or cantilever.

10. The bacterial nanocellulose composite of claim 1, wherein the nanocellulose comprises a surface or surface layer, wherein said surface or surface layer comprises sensor or signal processing molecule(s) (i) selected from: ion channel(s) or pore(s); membrane protein(s), lipoprotein(s), glycoproteins; receptor(s); enzymes, which are preferably active on the surface; or combinations thereof.

11. Use of a bacterial nanocellulose composite of claim 1 in material engineering, in chip technology, as printing matrix or printed nanocellulose composite, as transparent material or display or information processing device for LED and chips/chip technology, as printing, storage and/or processing medium, as detector, as intelligent foil, as intelligent material, as nanofactory, as sophisticated, light-controlled, synthesis device, as small biochemical analyzer, in DNA-based ASIC (application-specific chip) for sequence storage or analysis in wound healing and tissue engineering, as skin transplant, band-aid or tissue implant, as neuro transplant, for stimulus conduction, for muscle stimulation, as electronic skin, for monitoring wound healing, heartbeat, or other physical parameters, for faster regeneration, for reprogramming body cells during the healing process, or as an intelligent plaster.

12. (canceled)

13. The use according to claim 11, wherein the bacterial nanocellulose composite is used in a form of a hydrogel, a foil, a layer, or optical transparent paper.

14. An article of manufacture comprising the bacterial nanocellulose composite of claim 1 wherein said article of manufacture is selected from a printing, storage and/or processing medium; a smart card or a chip card; a skin transplant; a tissue implant: a neuro transplant and electronic skin.

15-16. (canceled)

17. A method for treating a wound, detecting a wound and/or monitoring wound healing wherein said method comprises the use of the bacterial nanocellulose composite of claim 1.

18. A method for tissue engineering wherein said method comprises the use of the bacterial nanocellulose composite of claim 1.

19. (canceled)

20. A method for stimulus conduction, muscle stimulation and/or monitoring a heartbeat, wherein said method comprises the use of bacterial nanocellulose composite of claim 1.

21. (canceled)

22. A method for producing a nanocellulose composite chip, comprising the steps of (1) providing a nanocellulose composite, preferably as defined in claim 1, (2) using a 3D printer or laser sintering, and (3) obtaining the nanocellulose composite chip.

23. The method of claim 22, wherein the nanocellulose in step (1) is bacterial nanocellulose, bacterial cellulose/poly caprolactone nanocomposite film, composite film of polyvinyl alcohol, bifunctional linking cellulose nanocrystals, or polylactide latex/nanofibrillated cellulose bio-nanocomposite, and/or wherein the 3D printer in step (2) is an ink-jet printer, a sinter printer, or a printer with melt layering.

24. A nanocellulose composite chip obtained by the method of claim 22.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0622] FIG. 1. Bacterial nanocellulose composite for information processing: use in chip technology.

[0623] A, Key components: Shown is a chip card made of bacterial nanocellulose (shown as fibers in the background) with embedded molecular switches (current- and signal-modulating pores, switch molecules (cylinders) or proteins having high resistance or capacitor characteristics, respectively (open squares).

[0624] B, In action: Nanocellulose composite containing information processing molecules (DNA/RNA polymerases or protein processing molecules) which may be controlled in their activity by different light wave lengths (top) by fusion to a light-sensing domain. Output is mediated by fluorescent proteins, actuator proteins, again in different wave-length. Membrane pores and modulation of membrane properties (optical, electronical properties of the nanocellulose surface) allows modulation of electronic properties and interfacing to electronic devices.

[0625] FIG. 2. Bacterial nanocellulose composite for information processing: use in tissue engineering.

[0626] Shown is an optimized tissue implant of bacterial nanocellulose (shown as fibers in the background) with embedded growth-promoting biomolecules (arrows) and mesenchymal stem cells (star). Further molecules may include monitoring (GFP) and sensor molecules to monitor inflammation and temperature.

[0627] FIG. 3. Bacterial nanocellulose composite key components: Achieving light-gated DNA input and outputlight-controlled phosphate transfer.

[0628] Measurement (top): assay for T4 kinase DNA elongation constructs using processed fluorescent oligonucleotides (Song and Zhao, 2009), for monitoring their activity; construct calculations to predict joined cooperative changes after Halabi et al. (2009) and Lee et al. (2008). The aim (bottom): construction of protein chimeras which transfer signals from the light harvesting BLUF domain to the effector domain, here polynucleotide kinase (PNK), to achieve on or off switching of effector activity.

[0629] FIG. 4. Bacterial nanocellulose composite key components: Achieving light-gated DNA input and outputlight directed PolyU polymerase.

[0630] Top: A histidine in the PolyU polymerase domain (PDB file shown: 4FH3) determines A or, in alternative position, U elongation (Lunde et al., 2012). The histidine 336 may be tilted by light to achieve rapid changes in substrate specificity according to user-specified sequences of As and Us. Bottom: Activity of the PolyU polymerase has again to be under light-control by fusion to a BLUF domain.

[0631] FIG. 5. Bacterial nanocellulose composite key components: Achieving light-gated DNA input and outputactive DNA storage design. Input (top): -DNA polymerase is used to achieve light-gated (light-specific BLUF domain/-DNA polymerase constructs for each nucleotide) and template free DNA synthesis.

[0632] Output (bottom): light-gated exonuclease constructs (triangles) are fused to specific nucleotide-binding domains (squares) and trigger different fluorescent proteins for readout.

[0633] FIG. 6. Active DNA storage in bacterial nanocellulose composite.

[0634] Previous efforts used living bacteria in a biofilm to achieve this storage (see DPA 10 2013 004 584.3). However, this can be difficult to manage, to maintain, to controlin particular, bacterial cells divide, need nutrients and escape by mutations control. The bacterial nanocellulose composite of the present invention solves all these problems and leads to a much more reliable, improved storage.

[0635] A, artificial biofilm blueprint for active multicomponent DNA storage: Each nanocellulose composite carries light-gated constructs for active DNA storage; input: light gated (L) BLUF domain B controls MU DNA polymerase constructs, four such constructs (4) write GATC nucleotides into DNA (D); regulatory light (L*) gated interface domain I; output: light-gated (L) exonuclease (Exo) together with nucleotide binding domain (NucB) directs fluorescent protein (FP) expression or signalling, again four different constructs are required. Furthermore, nanocellulose composite interconnections have to be modified by light-gated (Li, stippled arrows) opening of pores (for DNA PD or ion current P) to achieve controlled multi-cellular DNA storage and exchange as well as to achieve circuits with electronic properties.

[0636] B, Comparison: engineered patterns in a real biofilm: We show the high self-repair potential, the patterning of the biofilm, and restoration of biofilm formation potential. Readout is done here by different optical appearance; available are also different FP constructs and lacZ constructs. In the example (B. subtilis bacteria) key sensor histidine kinase genes were artificially deleted (kinC, kinD). This abolishes biofilm formation or any tight connections (see FIG. 6A) between cells (left colonies: no biofilm formed). There are spontaneous mutations in the strong biofilm repressor sin R which turn tight interaction back on and achieve patterning of colonies with biofilm forming and non-forming regions (right colony). Change in DNA content for all these specific mutations is actively monitored and visible. For large-scale active DNA storage it is highly advantageous to introduce the light gated and monitoring constructs in one or several nanocellulose composites (including various technical improvements compared to FIG. 6A described in other sections of this document).

[0637] C, close up looks on the engineered biofilm (scales are indicated, focus: patterned region).

[0638] FIG. 7. Key components of nanocellulose composite: comparing active T4 kinase readout to control condition.

[0639] A, Control base-line level.

[0640] B, Active T4 kinase readout.

[0641] FIG. 8. Nanocellulose composite imbedded molecular components: BLUF domain.

[0642] Shown is testing of PCR fragments and vector constructs. 800 bp Fragment of the BLUF construct, testing the AccI cut, which should and does cut of the fragment.

[0643] FIG. 9. Nanocellulose composite imbedded molecular components: Monitoring light gated control of enzyme function by GFP constructs.

[0644] A, Comparing BLUF-PNK-GFP, BLUF-GFP, GFP construct, Fluorescence in the dark. All three show fluorescence, the additional BLUF-domain enhances fluorescence.

[0645] B, Comparing BLUF-GFP, control and BLUF-PNK-GFP construct. UV plus daylight shows that the BLUF-GFP constructs respond with fluorescence under daylight.

[0646] FIG. 10. Nanocellulose composite imbedded molecular components: Creating light-gated nucleotide processing enzymes (demonstrated here for Cid1, a polyU RNA polymerase).

[0647] A, Verification of BLUF-coding sequence from the transfected bacteria (Rosetta strain) by PCR reaction.

[0648] B, Verification of BLUF-Cid1 (long) and BLUF-Cid1 (cut) from the transfected bacteria (M15 strain) by PCR reaction.

[0649] FIG. 11. Nanocellulose composite imbedded molecular components: Light-gated control of fluorescence.

[0650] Results of a BLUF-GFP construct. No blue light leads to inactive BLUF domain and hence far less fluorescence.

[0651] Shown are cultured bacteria in Lysogeny broth under UV.

[0652] A, negative control, only non-transfected E. coli in LB media,

[0653] B, positive control, induced E. coli with GFP cultured in 20 ml of media,

[0654] C, induced E. coli with BLUF-GFP construct in 20 ml of media,

[0655] D, lysate of non-induced E. coli (negative control),

[0656] E, lysate of E. coli with BLUF-GFP construct.

[0657] FIG. 12. Nanocellulose composite imbedded molecular components: Light-gated GFP monitoring construct is demonstrated.

[0658] Here we show light-gated (blue light mediate) control of GFP fluorescence.

[0659] The comparative study of different GFP expression in the BLUF-GFP construct under different conditions (magnification 100).

[0660] A, 16 hrs of cultivation in daylight (phase contrast),

[0661] B, 16 hrs of cultivation in daylight (under UV),

[0662] C, 16 hrs of cultivation in dark (phase contrast),

[0663] D, 16 hrs of cultivation in dark (under UV),

[0664] E, 24 hrs of cultivation in daylight (phase contrast),

[0665] F, 24 hrs of cultivation in daylight (under UV),

[0666] G, 24 hrs of cultivation in dark (phase contrast),

[0667] H, 24 hrs cultivation in dark (under UV).

[0668] FIG. 13. Nanocellulose composite imbedded molecular components: Light-gated RNA polymerase CidI.

[0669] A, Shown is SDS-PAGE with protein lysates of recombinant BLUF and BLUF-Cid1 constructs.

[0670] Lane 1marker, line 2BLUF-GFP, lane 3BLUF-Cid1 (cut), lane 4BLUF-Cid1 (long), lane 5BLUF-GFP, lane 6negative control, lysate from the non-induced cells.

[0671] B, Western-blot analysis of different BLUF constructs. Spot ABLUF-GFP, Spot 2BLUF-Cid1 (cut), spot 3BLUF-Cid1 (long).

[0672] FIG. 14. Nanocellulose composite: Nanocellulose generation.

[0673] Amplification of BcsA and BcsB. Line 1BcsA, line 2BcsB.

[0674] FIG. 15. Nanocellulose composite: Nanocellulose together with green or red reporters.

[0675] Visualization of E. coli transformed by BcsA/BcsB with fluorescent reporters.

[0676] A, BcsA protein fused with GFP,

[0677] B, BcsB protein fused with mCHERRY.

[0678] FIG. 16. Nanocellulose composite: Nanocellulose production.

[0679] Here time-dependent expression of rBcsB.

[0680] Lane 1whole cell lysate of BcsB, lane 2-6 hrs induction, lane 3-21 hrs induction, lane 4-30 hrs induction, lane 5-45 hrs induction. Arrow depicts the BcsB protein.

[0681] FIG. 17. Nanocellulose composite:

[0682] Nanocellulose stained by mCHERRY protein.

[0683] A and C, stained nanocellulose under red fluorescence.

[0684] B and D, the corresponding picture with phase contrast.

[0685] E and F, negative control, only nanocellulose (non-stained) under UV (E) with corresponding phase contrast (F).

[0686] FIG. 18. Nanocellulose composite:

[0687] Nanocellulose stained by GFP protein.

[0688] A, stained nanocellulose under green fluorescence.

[0689] B, corresponding picture with phase contrast.

[0690] C and D, negative control, only nanocellulose (non-stained) under UV (C) with corresponding phase contrast (D).

[0691] FIG. 19. Nanocellulose composite:

[0692] Flexible 3D Printer used in the 3D printing experiments for the nanocellulose composite.

[0693] FIG. 20. Detection of polymerase activity of light-gated RNA polymerase CidI with a molecular beacon assay.

[0694] A, shows design and structural parameters of molecular beacons.

[0695] B, shows that molecular beacons in solution can have three phases: bound to target, closed and random coil.

[0696] C and D, show the structure of the beacon MB1 alone (C) and with the oligonucleotide (D), so the beacon in the target bound state; also measured when the Klenow polymerase is active).

[0697] E and F, show the structure of the beacon MB2 alone and with the oligonucleotide (so beacon in the target bound state; also measured when the CidI polymerase is active).

[0698] FIG. 21. Plasmid constructs for bacterial expression of nanocellulose.

[0699] A, Ligated BcsA into the pQU-30-mCHERRY-GFP vector. After the digestion with BamHI and SalI, the mCHERRY-coding region was excised and replaced with BcsA-coding region.

[0700] B, Ligated BcsB into the pQU-30-mCHERRY-GFP vector. After the digestion with KpnI and BlpI, the GFP-coding region was excised and replaced with BcsB-coding region.

EXAMPLES

Example 1 Light-Gated Polymerases and Kinases and their Application for Active DNA Storage in Nanocellulose Composite

[0701] Light-gated proteins provide not only an important basis for neurogenetics, they are also very useful to achieve storage, recall and modification of nucleotide sequences for long-term information storage as DNA. We test here BLUF- and LOV-domain fusion constructs fused to Cid I polymerase and T4 polynucleotide kinase. Fusion constructs are established and validated for their sequence. The light-gating property is tested in fluorescence assays regarding nucleotide extension as well as by GFP expression regarding processivity. In conclusion, these constructs allow light-gated elongation of nucleotide sequences, either by phosphorylation or by polyuridylation. We describe further constructs and modifications and that the full functionality of an active DNA storage can be obtained.

[0702] 1. Materials and Methods

[0703] 1.1 Structural and Statistical Predictions

[0704] Calculation of engineered mutations were performed using the SCA MATLAB toolbox published by Ranganathan et al. (see Halabi et al, 2009).

[0705] All tested primer constructs for different BLUF domains, specifically:

[0706] Construct series ABLUF(cut)-Linker-Cid I

[0707] Construct series BCid I(A)-Linker-BLUF(cut)-Linker-Cid I(B)

[0708] Construct series CCid I-Linker-BLUF

[0709] In each series, different linkers were tested (see below).

[0710] 1.2. Molecular Cloning and Tests

[0711] For cloning, the pGEM-T easy Vector system (Promega Corp.) was used.

[0712] 2. Results

[0713] 2.1. Key Steps for Active DNA Storage

[0714] A direct connection from molecular processing in cells and DNA to technical computers is necessary to achieve speed and calculation potential. Electronic properties of DNA (Timper et al. 2012) are difficult to handle We disclose herein for linking DNA information processing to in silico processing step-by-step in an efficient way to light-gated proteins (Liu et al., 2012). Light-gated proteins allow (i) control of their own and other enzyme activities, (ii) gene expression and protein-protein interactions, as well as (iii) to achieve patterning and directing cell to cell communication and integration of circuits. Containment features control the high biological repair and replication potential of such biobricks (Shetty et al., 2008) which together achieve extremely robust active DNA storage technology without negative side-effects or uncontrolled risks.

[0715] FIGS. 3-6 demonstrate which critical steps need to be achieved and a blueprint of the design with experimental data: Light gated enzyme elongates or modifies DNA according to a signal (FIG. 3), Light gated polymerase synthesizes a new sequence according to light-signal (FIG. 4; inset: Crystal structure of RNA Poly U polymerase and the critical histidine which directs A or U incorporation, and could be tilted by light input). Light gated constructs achieving light-directed DNA synthesis and DNA-sequence readout via optical signals (FIG. 5). FIG. 6A sketches active DNA storage applying the constructs shown in FIG. 3-5 in a bacterial biofilm. FIGS. 6 B and C show self-repair potential and experimental results for an own engineered biofilm.

[0716] 2.2. Testing Principles of DNA Storage

[0717] See FIGS. 6A to C.

[0718] 3.3. Demonstrating Active DNA Storage Enzymes

[0719] In the following, the key steps for active DNA storage were all examined in detail. Different ways to achieve active, light gated nucleotide synthesis were compared (T4 Polynucleotide kinase and Cid I poly U polymerase) as well as different light-gated domains to control their activity (BLUF domain or LOV domain). Furthermore, monitoring of construct activity was either done indirectly (activity monitoring by fluorescent oligo in vitro after protein purification) or directly (activity monitoring of the construct within the bacteria by GFP construct). Furthermore, the resulting product is either tested biochemically (modification of the oligo), optically (fluorescence and modification by blue light) or by sequencing of the product.

[0720] We summarize in the following all different combinations tested and the evidence collected for the construct activities:

[0721] BLUF-T4 Polynucleotide kinase construct: Truncated BLUF domain with optimal length according to SCA analysis is fused to polynucleotide kinase. The PCR product was cloned in plasmids, expressed and verified and the protein purified. For details, see below.

[0722] Furthermore, control experiments measured T4 kinase activity using fluorescent oligos compared to negative controls.

[0723] b) Three different BLUF-Cid I constructs test control of Cid I polymerase activity by BLUF.

[0724] c) As an alternative, different LOV constructs test Cid I activity. These constructs perform similarly well, however, the required wave length for light-gating Cid I is different.

[0725] d) Direct monitoring within bacteria by GFP constructs. Constructs include: GFP alone, BLUF-GFP, BLUF-Cid I-GFP, BLUF-Cid I-BLUF-GFP.

[0726] Fluorescence is observed for the constructs and the BLUF domain controls Cid I as well as GFP activity.

[0727] The accession numbers for these different proteins and genes are as follows: [0728] Polynucleotide kinase gene (NC_000866 REGION: complement (134002 . . . 134907) from complete T4 genome) with polynucleotide kinase protein (accession number KJ477686.1) from enterobacteria phage T4. [0729] See SEQ ID NO. 20 (showing the 301 amino acid sequence of polynucleotide kinase of enterobacteria phage T4). [0730] Poly(A) polymerase Cid1 (accession number NP_594901) gene from Schizosaccharomyces pombe. [0731] See SEQ ID NO. 9 for the 405 amino acid sequence. [0732] The BLUF domain was obtained as part of the YcgF gene and protein (Tschwori et al., 2009; Tschwori et al., 2012). The used DNA for the BLUF domain was hence gene ycgF (accession number AAC74247.3) from the E. coli strain DH5-, amplicon from 1-375 nt (125 AA). See SEQ ID NOs. 1-7, wherein [0733] SEQ ID NO. 1 shows the 403 amino acid sequence of BLUF E. coli; [0734] SEQ ID NO. 2 shows aa 1-84 of SEQ ID NO. 1 and SEQ ID NO. 3 the respective nucleotide sequence; [0735] SEQ ID NO. 4 shows aa 1-144 of SEQ ID NO. 1 and SEQ ID NO. 5 the respective nucleotide sequence; [0736] SEQ ID NO. 6 shows aa 1-125 of SEQ ID NO. 1 and SEQ ID NO. 7 the respective nucleotide sequence.

[0737] The used DNA for the Cid1 construct started with poly(A) polymerase Cid1 (accession number NP_594901) from the yeast Schizosaccharomyces pombe [972h-]. [0738] See SEQ ID NO. 9 for the 405 amino acid sequence. [0739] SEQ ID NO. 10 shows aa 33-405 of SEQ ID NO. 9 and SEQ ID NO. 11 the respective nucleotide sequence; [0740] SEQ ID NO. 12 shows aa 1-377 of SEQ ID NO. 9 and SEQ ID NO. 13 the respective nucleotide sequence; [0741] SEQ ID NO. 14 shows aa 1-331 of SEQ ID NO. 9 and SEQ ID NO. 15 the respective nucleotide sequence; [0742] SEQ ID NO. 16 shows aa 332-405 of SEQ ID NO. 9 and SEQ ID NO. 17 the respective nucleotide sequence.

[0743] Different constructs used these proteins but modified the DNA encoding these to achieve an optimal construct for our purposes. In particular, Cid1 polymerase synthesizes poly U stretches, but can be modified to synthesize poly A (Lunde et al., 2012) and our novel constructs allow to switch the CidI activity on and off by having blue light exposure there or not.

[0744] As the sequences have been modified, the resulting nucleotide sequences are shown in the following.

[0745] 2.4 Polynucleotide Kinase (PKN)

[0746] The following construct was established: BLUF domain (Blue light responsive protein domain) is optimized in its length (so that it transmits cooperative changes) to T4 polynucleotide kinase. Such construct was compared to control conditions in a fluorescence monitoring assay of T4 polynucleotide kinase. FIG. 7 compares active T4 kinase readout to control condition. FIG. 11B shows that PKN-GFP or GFP alone can be controlled by blue light/day light using the BLUF domain.

[0747] 2.5. BLUF/Cid I Construct

[0748] The first construct attaches the predicted active part of a BLUF signalling protein (amino acids 1-84 of SEQ ID NO. 1) to a complete Cid I polymerase protein (amino acids 33-405 of SEQ ID NO. 9). The Cid I part is located at the C-terminal part of the designed fusion protein.

[0749] Construct ABLUF(cut)-Linker-Cid I

TABLE-US-00003 BLUF SEQIDNOs.2and3 MLTTLIYRSHIRDDEPVKKIEEMVSIANRRNMQSDVTGILLFNGSHFFQLLEGPEEQVKMIYRAICQDPRHYNIV ELLCDYAPA ApaIHindIII AAAAAA.GCGCGCGC.GGGCCC.AAGCTT. ATGCTTACCACCCTTATT ATGCTTACCACCCTTATTTATCGTAGCCATATACGTGACGACGAACCTGTCAAAAAAATCGAAGAAATGGTTTCG ATAGCAAATCGCAGGAACATGCAGTCTGACGTAACAGGGATCTTACTGTTTAATGGTTCTCATTTTTTCCAGCTT CTGGAAGGTCCGGAAGAACAGGTTAAAATGATATATCGGGCTATATGCCAGGATCCACGGCACTATAATATTGTT GAGCTGCTGTGCGATTACGCGCCTGCT TGCGATTACGCGCCTGCTGGTGGTGGTGGA TCCACCACCACCAGCAGGCGCGTAATCGCA Linker SEQIDNOs.18and19 GGGGSGGGGSGGGGS TACGCGCCTGCT GGTGGTGGTGGAAGCGGCGGCGGCGGCAGC GGTGGTGGTGGAAGCGGCGGCGGCGGCAGCGGCGGCGGAGGGAGC GGCGGCGGCGGCAGCGGCGGCGGAGGGAGCAGCTACCAAAAG CTTTTGGTAGCTGCTCCCTCCGCCGCCGCTGCCGCCGCCGCC CidI SEQIDNOs.10and11 SYQKVPNSHKEFTKFCYEVYNEIKISDKEFKEKRAALDTLRLCLKRISPDAELVAFGSLESGLALKNSDMDLCVL MDSRVQSDTIALQFYEELIAEGFEGKFLQRARIPIIKLTSDTKNGFGASFQCDIGFNNRLAIHNTLLLSSYTKLD ARLKPMVLLVKHWAKRKQINSPYFGTLSSYGYVLMVLYYLIHVIKPPVFPNLLLSPLKQEKIVDGFDVGFDDKLE DIPPSQNYSSLGSLLHGFFRFYAYKFEPREKVVTFRRPDGYLTKQEKGWTSATEHTGSADQIIKDRYILAIEDPF EISHNVGRTVSSSGLYRIRGEFMAASRLLNSRSYPIPYDSLFEEAPIPPRRQKKTDEQSNKKLLNETDGDNSE*S TOP GGCGGAGGGAGC AGCTACCAAAAGGTCCCT AGCTACCAAAAGGTCCCTAATTCGCACAAGGAATTTACGAAGTTTTGCTATGAAGTGTATAATGAGATTAAAATT AGTGACAAAGAGTTTAAAGAAAAGAGAGCGGCATTAGATACACTTCGGCTATGCCTTAAACGAATATCCCCTGAT GCTGAATTGGTAGCCTTTGGAAGTTTGGAATCTGGTTTAGCACTTAAAAATTCGGATATGGATTTGTGCGTGCTT ATGGATTCGCGCGTCCAAAGTGATACAATTGCGCTCCAATTCTATGAAGAGCTTATAGCTGAAGGATTTGAAGGA AAATTTTTACAAAGGGCAAGAATTCCCATTATCAAATTAACATCTGATACGAAAAATGGATTTGGGGCTTCGTTT CAATGTGATATTGGATTTAACAATCGTCTAGCTATTCATAATACGCTTTTACTTTCTTCATATACAAAATTAGAT GCTCGCCTAAAACCCATGGTCCTTCTTGTTAAGCATTGGGCCAAACGGAAGCAAATCAACTCTCCTTACTTTGGA ACTCTTTCCAGTTATGGTTACGTCCTAATGGTTCTTTACTATCTGATTCACGTTATCAAGCCTCCCGTCTTTCCT AATTTACTGTTGTCACCTTTGAAACAAGAAAAGATAGTTGATGGATTTGACGTTGGTTTTGACGATAAACTGGAA GATATCCCTCCTTCCCAAAATTATAGCTCATTGGGAAGTTTACTTCATGGCTTTTTTAGATTTTATGCTTATAAG TTCGAGCCACGGGAAAAGGTAGTAACTTTTCGTAGACCAGACGGTTACCTCACAAAGCAAGAGAAAGGATGGACT TCAGCTACTGAACACACTGGATCGGCTGATCAAATTATAAAAGACAGGTATATTCTTGCGATTGAAGATCCTTTC GAGATTTCACATAATGTGGGTAGGACAGTTAGCAGTTCTGGATTGTATCGGATTCGAGGGGAATTTATGGCCGCT TCAAGGTTGCTCAATTCTCGCTCATATCCTATCCCTTATGATTCATTATTTGAGGAGGCCCCAATTCCGCCTCGT CGCCAGAAAAAAACGGATGAACAATCTAACAAAAAATTGTTGAATGAAACCGATGGTGACAATTCTGAGTGA GGTGACAATTCTGAGTGA TTTTTT.CCGCGG.GGTACC.TCACTCAGAATTGTCACC SacIIKpnI

2.6. Cid I/BLUF Constructs

[0750] The second series of constructs is designed to insert the predicted active part of a BLUF signalling protein to the Cid I polymerase sequence. The locations for insertion were predicted to be functionally coupled to a Cid I polymerase activity regulating site.

[0751] Cid I(A) refers to amino acids 1-331 of SEQ ID NO. 9, and Cid I(B) refers to amino acids 332-405 of SEQ ID NO. 9.

TABLE-US-00004 ConstructB-CidI(A)-Linker-BLUF(cut)-Linker-CidI(B) CidI(A) SEQIDNOs.14and15 MNISSAQFIPGVHIVEEIEAEIHKNLHISKSCSYQKVPNSHKEFTKFCYEVYNEIKISDKEFKEKRAALDTLRLC LKRISPDAELVAFGSLESGLALKNSDMDLCVLMDSRVQSDTIALQFYEELIAEGFEGKFLQRARIPIIKLTSDTK NGFGASFQCDIGFNNRLAIHNTLLLSSYTKLDARLKPMVLLVKHWAKRKQINSPYFGTLSSYGYVLMVLYYLIHV IKPPVFPNLLLSPLKQEKIVDGFDVGFDDKLEDIPPSQNYSSLGSLLHGFFRFYAYKFEPREKVVTFRRPDGYLT KQEKGWTSATEHTGSADQIIKDRYILAIEDP ApaIHindIII AAAAAA.GCCCTT.GGGCCC.AAGCTT. ATGAACATTTCTTCTGCA ATGAACATTTCTTCTGCACAATTTATTCCTGGTGTTCACACAGTTGAAGAGATTGAGGCAGAAATTCACAAAAAT TTACATATTTCAAAAAGTTGTAGCTACCAAAAGGTCCCTAATTCGCACAAGGAATTTACGAAGTTTTGCTATGAA GTGTATAATGAGATTAAAATTAGTGACAAAGAGTTTAAAGAAAAGAGAGCGGCATTAGATACACTTCGGCTATGC CTTAAACGAATATCCCCTGATGCTGAATTGGTAGCCTTTGGAAGTTTGGAATCTGGTTTAGCACTTAAAAATTCG GATATGGATTTGTGCGTGCTTATGGATTCGCGCGTCCAAAGTGATACAATTGCGCTCCAATTCTATGAAGAGCTT ATAGCTGAAGGATTTGAAGGAAAATTTTTACAAAGGGCAAGAATTCCCATTATCAAATTAACATCTGATACGAAA AATGGATTTGGGGCTTCGTTTCAATGTGATATTGGATTTAACAATCGTCTAGCTATTCATAATACGCTTTTACTT TCTTCATATACAAAATTAGATGCTCGCCTAAAACCCATGGTCCTTCTTGTTAAGCATTGGGCCAAACGGAAGCAA ATCAACTCTCCTTACTTTGGAACTCTTTCCAGTTATGGTTACGTCCTAATGGTTCTTTACTATCTGATTCACGTT ATCAAGCCTCCCGTCTTTCCTAATTTACTGTTGTCACCTTTGAAACAAGAAAAGATAGTTGATGGATTTGACGTT GGTTTTGACGATAAACTGGAAGATATCCCTCCTTCCCAAAATTATAGCTCATTGGGAAGTTTACTTCATGGCTTT TTTAGATTTTATGCTTATAAGTTCGAGCCACGGGAAAAGGTAGTAACTTTTCGTAGACCAGACGGTTACCTCACA AAGCAAGAGAAAGGATGGACTTCAGCTACTGAACACACTGGATCGGCTGATCAAATTATAAAAGACAGGTATATT CTTGCGATTGAAGATCCT CTTGCGATTGAAGATCCTGGCGGCGGAGGG CCCTCCGCCGCCAGGATCTTCAATCGCAAG Linker SEQIDNOs.18and19 GGGGSGGGGSGGGGS ATTGAAGATCCT GGCGGCGGAGGGAGTGGTGGCGGAGGGTCA GGCGGCGGAGGGAGTGGTGGCGGAGGGTCAGGGGGCGGCGGCAGC GGTGGCGGAGGGTCAGGGGGCGGCGGCAGCATGCTTACCACC GGTGGTAAGCATGCTGCCGCCGCCCCCTGACCCTCCGCCACC BLUF SEQIDNOs.2and3 MLTTLIYRSHIRDDEPVKKIEEMVSIANRRNMQSDVTGILLFNGSHFFQLLEGPEEQVKMIYRAICQDPRHYNIV ELLCDYAPA GGCGGCGGCAGC ATGCTTACCACCCTTATT ATGCTTACCACCCTTATTTATCGTAGCCATATACGTGACGACGAACCTGTCAAAAAAATCGAAGAAATGGTTTCG ATAGCAAATCGCAGGAACATGCAGTCTGACGTAACAGGGATCTTACTGTTTAATGGTTCTCATTTTTTCCAGCTT CTGGAAGGTCCGGAAGAACAGGTTAAAATGATATATCGGGCTATATGCCAGGATCCACGGCACTATAATATTGTT GAGCTGCTGTGCGATTACGCGCCTGCT TGCGATTACGCGCCTGCTGGAGGAGGAGGA TCCTCCTCCTCCAGCAGGCGCGTAATCGCA Linker SEQIDNOs.18and19 GGGGSGGGGSGGGGS TACGCGCCTGCT GGAGGAGGAGGATCCGGGGGAGGCGGTTCT GGAGGAGGAGGATCCGGGGGAGGCGGTTCTGGCGGCGGGGGCAGC GGGGGAGGCGGTTCTGGCGGCGGGGGCAGCTTCGAGATTTCA TGAAATCTCGAAGCTGCCCCCGCCGCCAGAACCGCCTCCCCC CidI(B) SEQIDNOs.16and17 FEISHNVGRTVSSSGLYRIRGEFMAASRLLNSRSYPIPYDSLFEEAPIPPRRQKKTDEQSNKKLLNETDGDNSE* STOP GGCGGGGGCAGC TTCGAGATTTCACATAAT TTCGAGATTTCACATAATGTGGGTAGGACAGTTAGCAGTTCTGGATTGTATCGGATTCGAGGGGAATTTATGGCC GCTTCAAGGTTGCTCAATTCTCGCTCATATCCTATCCCTTATGATTCATTATTTGAGGAGGCCCCAATTCCGCCT CGTCGCCAGAAAAAAACGGATGAACAATCTAACAAAAAATTGTTGAATGAAACCGATGGTGACAATTCTGAGTGA GGTGACAATTCTGAGTGA TTTTTT.CCGCGG.GGTACC.TCACTCAGAATTGTCACC SacIIKpnI

[0752] 2.7. Cid I/BLUF (Complete) Construct

[0753] The third construct is designed for verification. The domain assembly is reversed in comparison to the first two series: Cid I polymerase (amino acids 1-377 of SEQ ID NO. 9) is located at the N-terminal part, while BLUF makes the C-terminus of the fusion protein. Both domains feature unedited complete sequences.

[0754] Construct CCid I-Linker-BLUF

TABLE-US-00005 CidI SEQIDNOs.12and13 MNISSAQFIPGVHTVEEIEAEIHKNLHISKSCSYQKVPNSHKEFTKFCYEVYNEIKISDKEFKEKRAALDTLRLC LKRISPDAELVAFGSLESGLALKNSDMDLCVLMDSRVQSDTIALQFYEELIAEGFEGKFLQRARIPIIKLTSDTK NGFGASFQCDIGFNNRLAIHNTLLLSSYTKLDARLKPMVLLVKHWAKRKQINSPYFGTLSSYGYVLMVLYYLIHV IKPPVFPNLLLSPLKQEKIVDGFDVGFDDKLEDIPPSQNYSSLGSLLHGFFRFYAYKFEPREKVVTFRRPDGYLT KQEKGWTSATEHTGSADQIIKDRYILAIEDPFEISHNVGRTVSSSGLYRIRGEFMAASRLLNSRSYPIPYDSLFE EA ApaIHindIII AAAAAA.GGGCCC.AAGCTT. ATGAACATTTCTTCTGCA ATGAACATTTCTTCTGCACAATTTATTCCTGGTGTTCACACAGTTGAAGAGATTGAGGCAGAAATTCACAAAAAT TTACATATTTCAAAAAGTTGTAGCTACCAAAAGGTCCCTAATTCGCACAAGGAATTTACGAAGTTTTGCTATGAA GTGTATAATGAGATTAAAATTAGTGACAAAGAGTTTAAAGAAAAGAGAGCGGCATTAGATACACTTCGGCTATGC CTTAAACGAATATCCCCTGATGCTGAATTGGTAGCCTTTGGAAGTTTGGAATCTGGTTTAGCACTTAAAAATTCG GATATGGATTTGTGCGTGCTTATGGATTCGCGCGTCCAAAGTGATACAATTGCGCTCCAATTCTATGAAGAGCTT ATAGCTGAAGGATTTGAAGGAAAATTTTTACAAAGGGCAAGAATTCCCATTATCAAATTAACATCTGATACGAAA AATGGATTTGGGGCTTCGTTTCAATGTGATATTGGATTTAACAATCGTCTAGCTATTCATAATACGCTTTTACTT TCTTCATATACAAAATTAGATGCTCGCCTAAAACCCATGGTCCTTCTTGTTAAGCATTGGGCCAAACGGAAGCAA ATCAACTCTCCTTACTTTGGAACTCTTTCCAGTTATGGTTACGTCCTAATGGTTCTTTACTATCTGATTCACGTT ATCAAGCCTCCCGTCTTTCCTAATTTACTGTTGTCACCTTTGAAACAAGAAAAGATAGTTGATGGATTTGACGTT GGTTTTGACGATAAACTGGAAGATATCCCTCCTTCCCAAAATTATAGCTCATTGGGAAGTTTACTTCATGGCTTT TTTAGATTTTATGCTTATAAGTTCGAGCCACGGGAAAAGGTAGTAACTTTTCGTAGACCAGACGGTTACCTCACA AAGCAAGAGAAAGGATGGACTTCAGCTACTGAACACACTGGATCGGCTGATCAAATTATAAAAGACAGGTATATT CTTGCGATTGAAGATCCTTTCGAGATTTCACATAATGTGGGTAGGACAGTTAGCAGTTCTGGATTGTATCGGATT CGAGGGGAATTTATGGCCGCTTCAAGGTTGCTCAATTCTCGCTCATATCCTATCCCTTATGATTCATTATTTGAG GAGGCC TCATTATTTGAGGAGGCCGGAGGAGGAGGT ACCTCCTCCTCCGGCCTCCTCAAATAATGA Linker SEQIDNOs.18and19 GGGGSGGGGSGGGGS TTTGAGGAGGCC GGAGGAGGAGGTAGCGGTGGCGGAGGGTCA GGAGGAGGAGGTAGCGGTGGCGGAGGGTCAGGTGGCGGCGGGAGT GGTGGCGGAGGGTCAGGTGGCGGCGGGAGTATGCTTACCACC GGTGGTAAGCATACTCCCGCCGCCACCTGACCCTCCGCCACC BLUF SEQIDNOs.4and5 MLTTLIYRSHIRDDEPVKKIEEMVSIANRRNMQSDVTGILLFNGSHFFQLLEGPEEQVKMIYRAICQDPRHYNIV ELLCDYAPARRFGKAGMELFDLRLHERDDVLQAVFDKGTSKFQLTYDDRALQFFRTFVLATEQSTYFEI*STOP GGCGGCGGGAGT ATGCTTACCACCCTTATT ATGCTTACCACCCTTATTTATCGTAGCCATATACGTGACGACGAACCTGTCAAAAAAATCGAAGAAATGGTTTCG ATAGCAAATCGCAGGAACATGCAGTCTGACGTAACAGGGATCTTACTGTTTAATGGTTCTCATTTTTTCCAGCTT CTGGAAGGTCCGGAAGAACAGGTTAAAATGATATATCGGGCTATATGCCAGGATCCACGGCACTATAATATTGTT GAGCTGCTGTGCGATTACGCGCCTGCTCGCCGTTTTGGCAAAGCGGGAATGGAATTATTTGATTTGCGCCTGCAC GAGCGAGATGACGTTTTACAGGCCGTATTCGACAAAGGCACATCAAAATTTCAGCTAACTTATGATGACAGAGCG CTACAATTTTTTCGTACTTTTGTCCTTGCAACCGAACAATCAACCTATTTCGAGATCTAA ACCTATTTCGAGATCTAA TTTTTT.TCT.CCGCGG.GGTACC.TTAGATCTCGAAATAGGT SacIIKpnI

[0755] 2.8. BLUF/Cid I/GFP (Preparation) Construct

[0756] The fourth series of constructs is designed to insert the predicted active part of a BLUF signalling protein to the Cid I polymerase sequence. To add an additional internal control mechanism a second BLUF domain together with a linker structure is attached to GFP. The second BLUF domain is located at the C-terminus of the resulting fusion protein and prepares expression in a GFP-containing expression vector system. The GFP domain sequence is already integrated into the chosen expression vector system.

[0757] Construct DBLUF(Cut)-Linker 1-Cid I-Linker 2-BLUF(Cut Long)-GFP(Prepare)

TABLE-US-00006 BLUF SEQIDNOs.2and3 MLTTLIYRSHIRDDEPVKKIEEMVSIANRRNMQSDVTGILLFNGSHFFQLLEGPEEQVKMIYRAICQDPRHYNIV ELLCDYAPA PstIBglII AAAAAA.CGCGCGCGC.CTGCAG.AGATCT. ATGCTTACCACCCTTATT ATGCTTACCACCCTTATTTATCGTAGCCATATACGTGACGACGAACCTGTCAAAAAAATCGAAGAAATGGTTTCG ATAGCAAATCGCAGGAACATGCAGTCTGACGTAACAGGGATCTTACTGTTTAATGGTTCTCATTTTTTCCAGCTT CTGGAAGGTCCGGAAGAACAGGTTAAAATGATATATCGGGCTATATGCCAGGATCCACGGCACTATAATATTGTT GAGCTGCTGTGCGATTACGCGCCTGCT TGCGATTACGCGCCTGCTGGTGGTGGTGGT ACCACCACCACCAGCAGGCGCGTAATCGCA Linker SEQIDNOs.18and19 GGGGSGGGGSGGGGS TACGCGCCTGCT GGTGGTGGTGGTTCTGGTGGTGGTGGTAGT GGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGCGGAGGAGGGAGC GGTGGTGGTGGTAGTGGCGGAGGAGGGAGCAGCTACCAAAAG CTTTTGGTAGCTGCTCCCTCCTCCGCCACTACCACCACCACC CidI SEQIDNOs.10and11 SYQKVPNSHKEFTKFCYEVYNEIKISDKEFKEKRAALDTLRLCLKRISPDAELVAFGSLESGLALKNSDMDLCVL MDSRVQSDTIALQFYEELIAEGFEGKFLQRARIPIIKLTSDTKNGFGASFQCDIGFNNRLAIHNTLLLSSYTKLD ARLKPMVLLVKHWAKRKQINSPYFGTLSSYGYVLMVLYYLIHVIKPPVFPNLLLSPLKQEKIVDGFDVGFDDKLE DIPPSQNYSSLGSLLHGFFRFYAYKFEPREKVVTFRRPDGYLTKQEKGWTSATEHTGSADQIIKDRYILAIEDPF EISHNVGRTVSSSGLYRIRGEFMAASRLLNSRSYPIPYDSLFEEAPIPPRRQKKTDEQSNKKLLNETDGDNSE*S TOP GGAGGAGGGAGC AGCTACCAAAAGGTCCCT AGCTACCAAAAGGTCCCTAATTCGCACAAGGAATTTACGAAGTTTTGCTATGAAGTGTATAATGAGATTAAAATT AGTGACAAAGAGTTTAAAGAAAAGAGAGCGGCATTAGATACACTTCGGCTATGCCTTAAACGAATATCCCCTGAT GCTGAATTGGTAGCCTTTGGAAGTTTGGAATCTGGTTTAGCACTTAAAAATTCGGATATGGATTTGTGCGTGCTT ATGGATTCGCGCGTCCAAAGTGATACAATTGCGCTCCAATTCTATGAAGAGCTTATAGCTGAAGGATTTGAAGGA AAATTTTTACAAAGGGCAAGAATTCCCATTATCAAATTAACATCTGATACGAAAAATGGATTTGGGGCTTCGTTT CAATGTGATATTGGATTTAACAATCGTCTAGCTATTCATAATACGCTTTTACTTTCTTCATATACAAAATTAGAT GCTCGCCTAAAACCCATGGTCCTTCTTGTTAAGCATTGGGCCAAACGGAAGCAAATCAACTCTCCTTACTTTGGA ACTCTTTCCAGTTATGGTTACGTCCTAATGGTTCTTTACTATCTGATTCACGTTATCAAGCCTCCCGTCTTTCCT AATTTACTGTTGTCACCTTTGAAACAAGAAAAGATAGTTGATGGATTTGACGTTGGTTTTGACGATAAACTGGAA GATATCCCTCCTTCCCAAAATTATAGCTCATTGGGAAGTTTACTTCATGGCTTTTTTAGATTTTATGCTTATAAG TTCGAGCCACGGGAAAAGGTAGTAACTTTTCGTAGACCAGACGGTTACCTCACAAAGCAAGAGAAAGGATGGACT TCAGCTACTGAACACACTGGATCGGCTGATCAAATTATAAAAGACAGGTATATTCTTGCGATTGAAGATCCTTTC GAGATTTCACATAATGTGGGTAGGACAGTTAGCAGTTCTGGATTGTATCGGATTCGAGGGGAATTTATGGCCGCT TCAAGGTTGCTCAATTCTCGCTCATATCCTATCCCTTATGATTCATTATTTGAGGAGGCCCCAATTCCGCCTCGT CGCCAGAAAAAAACGGATGAACAATCTAACAAAAAATTGTTGAATGAAACCGATGGTGACAATTCTGAGTGA GGTGACAATTCTGAGTGAGGCGGAGGAGGT ACCTCCTCCGCCTCACTCAGAATTGTCACC Linker SEQIDNOs.18and19 GGGGSGGGGSGGGGS AATTCTGAGTGA GGCGGAGGAGGTAGCGGTGGCGGAGGGTCA GGCGGAGGAGGTAGCGGTGGCGGAGGGTCAGGTGGTGGGGGAAGT GGTGGCGGAGGGTCAGGTGGTGGGGGAAGTATGCTTACCACC GGTGGTAAGCATACTTCCCCCACCACCTGACCCTCCGCCACC BLUF SEQIDNOs.6and7 MLTTLIYRSHIRDDEPVKKIEEMVSIANRRNMQSDVTGILLENGSHFFQLLEGPEEQVKMIYRAICQDPRHYNIV ELLCDYAPARRFGKAGMELFDLRLHERDDVLQAVFDKGTSKFQLTYDDRA GGTGGGGGAAGT ATGCTTACCACCCTTATT ATGCTTACCACCCTTATTTATCGTAGCCATATACGTGACGACGAACCTGTCAAAAAAATCGAAGAAATGGTTTCG ATAGCAAATCGCAGGAACATGCAGTCTGACGTAACAGGGATCTTACTGTTTAATGGTTCTCATTTTTTCCAGCTT CTGGAAGGTCCGGAAGAACAGGTTAAAATGATATATCGGGCTATATGCCAGGATCCACGGCACTATAATATTGTT GAGCTGCTGTGCGATTACGCGCCTGCTCGCCGTTTTGGCAAAGCGGGAATGGAATTATTTGATTTGCGCCTGCAC GAGCGAGATGACGTTTTACAGGCCGTATTCGACAAAGGCACATCAAAATTTCAGCTAACTTATGATGACAGAGCG ACTTATGATGACAGAGCG AAAAAA.CTCGAG.AAGCTT.CGCTCTGTCATCATAAGT XhoIHindIII

[0758] 2.9. BLUF/GFP (Preparation) Construct

[0759] The fifth series of constructs is designed to insert the predicted active part of a BLUF signalling protein to the GFP reporter domain sequence. While BLUF makes the N-terminus of the fusion protein, the GFP domain sequence is already integrated into the chosen expression vector system.

[0760] The predicted change in GFP activity level is shown in FIG. 9.

[0761] Construct EBLUE (Cut)-GFP (Prepare)

TABLE-US-00007 BLUF SEQIDNOs.2and3 MLTTLIYRSHIRDDEPVKKIEEMVSIANRRNMQSDVTGILLFNGSHFFQLLEGPEEQVKMIYRAICQDPRHYNIV ELLCDYAPA PstIBglII AAAAAA.CTGCAG.AGATCT. ATGCTTACCACCCTTATTTATCGTAGC ATGCTTACCACCCTTATTTATCGTAGCCATATACGTGACGACGAACCTGTCAAAAAAATCGAAGAAATGGTTTCG ATAGCAAATCGCAGGAACATGCAGTCTGACGTAACAGGGATCTTACTGTTTAATGGTTCTCATTTTTTCCAGCTT CTGGAAGGTCCGGAAGAACAGGTTAAAATGATATATCGGGCTATATGCCAGGATCCACGGCACTATAATATTGTT GAGCTGCTGTGCGATTACGCGCCTGCTCGCCGTTTTGGCAAAGCGGGAATGGAATTATTTGATTTGCGCCTGCAC GAGCGAGATGACGTTTTACAGGCCGTATTCGACAAAGGCACATCAAAATTTCAGCTAACTTATGATGACAGAGCG TTTCAGCTAACTTATGATGACAGAGCG AAAAAA.CTCGAG.AAGCTT.CGCTCTGTCATCATAAGTTAGCTGAAA XhoIHindIII

Example 2 Bacterial Expression of BLUF-GFP and BLUF-Cid Constructs

[0762] BLUF-domain (the sensor for Blue Light Using FAD) is a novel blue light photoreceptor, identified in 2002 and it is found in more than 50 different proteins. These proteins are involved in various functions, such as photophobic responses (e.g. PAC proteinEuglena gracilis, Slr1694Synechocystis sp.) and regulation of transcription (e.g. AppA protein Rhodobacter sphaeroides, BlrpE. coli). The proteins containing BLUF or similar domain are also found in Klebsiella pneumoniae, Naegleria gruberi, Acinetobacter baylyi and many other organisms. The molecular mechanism of BLUF-domain is very sophisticated. It converts the light signal to the biological information, following the conformational changes of the photoreceptor. Those changes are then recognized by other protein modules that traverse the signal to the downstream machineries. This type of light signal transduction mechanism was specifically modified in each organism during the evolution, to allow the adaptation for the different environmental conditions.

[0763] Main Aim:

[0764] To produce BLUF and BLUF-Cid1 in E. coli expression system. See FIG. 10.

[0765] Tools:

[0766] A circular DNA plasmid pPK-CMV-F1 vector with inserted BLUF domain with GFP on C-terminus (BLUF-GFP construct, see FIG. 11)/A circular DNA plasmid pUC57 with inserted BLUF domain with Cid1 polymerase in short or long version [BLUF-Cid1(cut) construct, BLUF-Cid1(long) construct, see FIG. 13]. Between the sequence of BLUF and Cid1 in both constructs was used linker GGGGS GGGGS GGGGS, which does not affect the folding of the fusion protein partners.

[0767] Used DNA for the BLUF: gene ycgF (accession number AAC74247.3, see SEQ ID NO. 1.) from the E. coli strain DH5-,

[0768] amplicon from 1-375 nt (125 AA), see SEQ ID NOs. 6 and 7.

[0769] Used DNA for the Cid1: poly(A) polymerase Cid1 (accession number NP_594901) from the Schizosaccharomyces pombe [972h-]

[0770] SEQ ID NO. 9.

[0771] Used Primers:

[0772] Constructs BLUF-GFP:

[0773] BLUF-GFP FW:

TABLE-US-00008 SEQIDNO.20 AAAAAACTGCAGAGATCTATGCTTACCACCCTTATTTATCGTAGC

[0774] (including restriction sites for PstI and BglII)

[0775] BLUF-GFP RV:

TABLE-US-00009 SEQIDNO.21 TTTTTTGAGCTCTTCGAAGCGAGACAGTAGTATTCAATCGACTTT

[0776] (including restriction sites for XhoI and HindIII)

[0777] After amplification of BLUF domain, PCR product was digested and ligated into the pPK-CMV-F1 vector and ligation mix was used for the transfection of bacteria. As a host strain E. coli strain DH5- and E. coli strain Rosetta (chemical transformation) were used.

[0778] For the preparation of the BLUF-Cid1 constructs, the commercial service (GenScript) was used to prepare the vectors with inserted sequences. The plasmids were used for chemical transformation of E. coli strain M15.

[0779] After transformation, bacteria with BLUF-GFP construct were cultured in Lysogeny broth (LB) and the protein was expressed using 1 mM Isopropyl -D-1-thiogalactopyranoside (IPTG) (FIGS. 12 C and E). As a positive control, bacteria transformed with GFP only were used (FIG. 12 B). As a negative control, non-induced bacteria were used (FIGS. 12 C and D).

[0780] To assess whether the GFP is expressed under the control of BLUF domain, bacteria were cultivated under two different conditions (in dark or in light) for 16 and 24 hours with 1 mM IPTG on LB agar with selective antibiotics. The fluorescence of live bacteria was visualized with a fluorescent microscope. The results suggest that after 16 hours of incubation, the bacteria were fluorescent under the light conditions, but not the dark conditions (FIGS. 12 B and D). After 24 hours, the difference between the intensity of fluorescence under light or dark condition was not significant (FIG. 4, panel F and H).

[0781] Subsequently, the bacteria were harvested and lysed under the native conditions with native lysis buffer with 1 mg/ml of lysozyme and protease inhibitor cocktail, with short sonification (310 sec cycles). The cell debris was removed by centrifugation and the supernatant contains proteins were separated by PAGE under reducing conditions. As seen in FIG. 13 A, all the recombinant proteins were overexpressed. The BLUF domain itself was observed as a low-molecular weight protein (FIG. 13 A, lanes 2 and 6), while BLUF-Cid1 constructs were observed as a low-molecular weight component with MW approximately 13 kDa and two high-MW components, approximately 45 kDa and 58 kDa (FIG. 13 A, lanes 3 and 4). Predicted molecular weight for recombinant BLUF domain fragment is 10.2 kDa, for the Cid1 fragment 42.7 kDa and for the BLUF-Cid1 construct 54.2 kDa (predicted by Geneious software).

[0782] The BLUF-Cid1 construct contains also the HIS-tag for easier purification, whereas the vector containing BLUF-GFP insert did not contain any tag. Accordingly, the presence of the BLUF-Cid1 construct in the lysate was also detected by western blot. In short, the lysate of BLUF-GFP (as a negative control), BLUF-Cid1 (cut) and BLUF-Cid1(long) was trickled onto the nitrocellulose membrane, and after drying, the membrane was blocked with 2% bovine albumin to remove the non-specific interactions. Subsequently, membrane was hybridized with Ni-HRP conjugate and the presence of His-tagged proteins were visualized. In the case of BLUF-GFP, any protein was detected (FIG. 13 B, spot 1), while both constructs BLUF-Cid1 was detected (FIG. 13 B, spots 2 and 3).

Example 3 Detection of Polymerase Activity of BLUF-CidI

[0783] The GFP control construct series allows monitoring differences in activity for expressed fusion proteins. While the GFP control vector shows fluorescence activity at the expected standard level, the BLUF-GFP fusion constructs feature elevated activity levels both at UV lighting (FIG. 9A) and a combination of UV and daylight (FIG. 9B).

[0784] Detailed functional proof of the observed correct fluorescence activity of the constructs requires polymerase or kinase activity monitoring using a fluorescent oligonucleotide (FIG. 5). This was achieved for T4 polynucleotide kinase.

[0785] In addition, this was achieved with different Cid I polymerase constructs, [0786] i.e. synthesis of different nucleotide sequences and control by BLUF domains; confirmation for correctly synthesized sequences after switching the construct to on using molecular beacons.

[0787] as well as direct monitoring of fluorescence in read-out BLUF-GFP constructs [0788] i.e. switching off the BLUF domain by blue light stops then fluorescence; documented by light microscopy.

[0789] 1. Molecular Beacon Assay

[0790] The molecular beacon uses for CidI Polymerase activity monitoring an RNA beacon as template, the synthesized polyU from the light-gated activated (by blue light) Cid I polymerase opens up the beacon structure and fluorescence changes. Molecular beacons are advantageous in many applications to detect nucleic acid synthesis and quantify it. The stem-loop structure of a molecular beacon may open up or change and provides a competing reaction for probe-target hybridization. FIGS. 20 A and B are drawn and given according to Tsourkas et al., 2003 and illustrate the general technique: FIG. 20 A shows design and structural parameters of molecular beacons. FIG. 20 B shows that molecular beacons in solution can have three phases: bound to target, closed and random coil.

[0791] We then generated the following beacons and oligonucleotides/primers with fluorophor TAMRA and the quencher BHQ2 for our experiments:

[0792] 3.1 Control Experiments and Positive Controls Using DNA as Well as Klenow Fragment:

[0793] Beacon MB_1 (DNA):

TABLE-US-00010 SEQIDNO.23 5-TAMRA-CCTCGTGTCTTGTACTTCCCGTCCGAGG-BHQ2-3

[0794] This required the corresponding Oligo_A (DNA):

TABLE-US-00011 SEQIDNO.24 5-GACGGGAAGTACAAGACAC-3

[0795] For the experiments with the Klenow-fragment, the primer Oligo_B (DNA) was used:

TABLE-US-00012 5-GACGGGAAG-3

[0796] 3.2 for the Experiments with the Cid1-Poly-U-Polymerase we Used the Following Oligonucleotides:

[0797] Beacon MB_2_Poly-U (DNA):

TABLE-US-00013 SEQIDNO.25 5-TAMRA-CCTCAAAAAAAAAAAAAAACGCGGCTGAGG-BHQ2-3

[0798] With the corresponding oligonucleotide to open the beacon (positive control) Oligo_PUr (RNA):

TABLE-US-00014 SEQIDNO.26 5-GCCGCGUUUUUUUUUUUUUUU-3

[0799] For the activity monitoring of the Cid1-Polymerase, the primer Oligo_PriUr (RNA) was used:

TABLE-US-00015 SEQIDNO.27 5-GCCGCGUUUU-3

[0800] FIGS. 20 C and D show the structure of the beacon MB1 alone (C) and with the oligonucleotide (D), so the beacon in the target bound state; also measured when the Klenow polymerase is active).

[0801] FIGS. 20 E and F show the structure of the beacon MB2 alone and with the oligonucleotide (so beacon in the target bound state; also measured when the CidI polymerase is active).

[0802] We hence generated a molecular beacon for CidI polymerase activity monitoring so that it works and opens up to bind to the target as soon as there is polyU synthesized by the CidI polymerase, and then quencher and fluorophore are separated. In several independent experiments efficient polyU synthesis was observed only if the CidI polymerase construct was switched on and active. Moreover, this could only be observed for the blue-light gated form of the CidI polymerase construct when blue light was there and stopped, when the blue-light was switched off

[0803] 2. Light Microscopy Test

[0804] Another example is direct monitoring of fluorescence in read-out BLUF-GFP constructs i.e. switching off the BLUF domain by blue light stops then fluorescence; documented by light microscopy (FIG. 12).

[0805] Here one of the imbedded molecular components was tested, using a light-gated GFP monitoring construct. There is light-gated (blue light mediated) control of GFP fluorescence. The different panels show in detail how only blue light/daylight allows full GFP fluorescence to develop whereas no switching on of the blue light mediating BLUF domain strongly reduces obtained GFP fluorescence.

Example 4 Bacterial Expression of Nanocellulose

[0806] Nanocellulose is an emerging multipurpose biomaterial, which can be obtained from the two natural sources: from wood or microorganisms. The wooden nanocellulose is made from wood pulp, from which the non-cellulose components are removed. The purified pulp is then homogenized and the mixture is separated to cellulose fibers, which are then formed to paste, crystals or spaghetti-like fibers. Bacterial nanocellulose for the industrial and medical usage is prepared mostly by fermentation of Gluconacetobacter xylinus, but there are more species able to produce the cellulose, such as Achromobacter, Sarcina, Pseudomonas and Dickeya. Bacterial nanocellulose has several interesting features, such as unique nanostructure, high capacity to absorb water, high level of polymerization, followed by high mechanical strength and crystallinity, which categorize the nanocellulose to the group of potential ecological material for the 21th century.

[0807] Nanocellulose can be used in various fields of industry; pharmaceutical, food production, textile, electronic, cosmetic and many more areas.

[0808] The recombinant DNA technology is routinely used in agriculture, food industry and medicine, but currently there is a new challengeto produce the new biomaterials with desired properties. The materials, which have their origin in nature but are used in bioengineering are called recombinamers and we believe, that bacterial nanocellulose can be produced also in this manner.

[0809] Main Aim:

[0810] To produce nanocellulose in E. coli expression system.

[0811] Tools:

[0812] A circular DNA plasmid pQE-30-mCHERRY-GFP vector with inserted BcsA/BcsB unit, see FIGS. 21 A and B.

[0813] Used DNA for BcsA: gene bcsACellulose synthase catalytic subunit [UDP-forming] (accession number AAB18510.1.) from the E. coli strain DH5-11, amplicon from 34-2610 nt (858 AA).

[0814] See SEQ ID NO. 32 for the full length amino acid sequence (872 aa), as shown in Database: UniProt/SWISS-PROT, Entry: BCSA_ECOLI.

[0815] Used DNA for the BcsB: gene bcsBCyclic di-GMP-binding protein (accession number AAB18509.1.) from the E. coli strain DH5-, amplicon from 82-2331 nt (750 AA). See SEQ ID NO. 33 for the full length amino acid sequence (779 aa), as shown in Database: UniProt/SWISS-PROT, Entry: BCSB_ECOLI.

[0816] Used Primers:

[0817] BcsA-GFP Construct:

[0818] BcsA F:

TABLE-US-00016 SEQIDNO.28 TATGGATCCCCGGTCAACGCGCGGCTTATC

[0819] (including restriction site for BamHI)

[0820] BcsA R:

TABLE-US-00017 SEQIDNO.29 TCTGTCGACAGCCAAAGCCTGATCCGATGG

[0821] (including restriction site for SalI)

[0822] BcsB-mCHERRY Construct

[0823] BcsB F:

TABLE-US-00018 SEQIDNO.30 CCTGGTACCGCAACGCAACCACTGATCAAT

[0824] (including restriction site for KpnI)

[0825] BcsB R:

TABLE-US-00019 SEQIDNO.31 ATGCTCAGCATCCGGGTTAAGACGACGACG

[0826] (including restriction site for BlpI)

[0827] After amplification of BcsA and BcsB coding sequences (FIG. 14), PCR products was digested and ligated into the pQE-30 GFP or pQE-30 mCHERRY plasmid and ligation mixes were used for the transfection of bacteria. As a host strain was used E. coli strain M15 (chemical transformation).

[0828] After transformation, bacteria with constructs were cultured LB media and the proteins were expressed using 1 mM IPTG. The expressed proteins were visualized by fluorescent microscope. The bacteria with the construct BcsA emitted green fluorescence (FIG. 15 A), while bacteria transfected by BcsB red fluorescence (FIG. 15 B).

[0829] Transfected bacteria were after time-dependent induction (6, 21, 30 and 45 hrs) harvested and lysed under the denaturating conditions using 8M urea and purified by Ni-NTA resin. The BcsA construct was probably cleaved during the lysis so we didn't get any results on PAGE (predicted MW for the BcsA is 99.7 kDa, BcsA-GFP130 kDa, GFP30 kDa), but the BcsB was significantly overexpressed (predicted MW for the BcsB is 86 kDa, BcsB-mCHERRY112 kDa, mCHERRY 26 kDa) (FIG. 16). As the further experiment, BcsA and BcsB will be fused with overlapping primer to obtain one molecule with His-tag, without the fluorescent reporter.

[0830] As an initial experiment to test the properties of bacterial nanocellulose, we tried to prepare the fluorescent nanocellulose. Bacterial nanocellulose was kindly provided by Dr. Kralish (JeNaCell, Germany). The recombinant protein mCHERRY-GFP was prepared from the in-house modified plasmid pQE-30 GFP-mCHERRY and after purification was hybridized with nanocellulose for 24 hrs in 4 C. Fluorescence was asses by fluorescence microscope (100, FIG. 17, FIG. 18).

Example 5 3D Printing

[0831] We furthermore can show that our nanocellulose composite with all its components is a suitable object to be produced by 3D printing technology.

[0832] A standard printer can be used for this, however, as is known for 3D printing of biological objects such as tissues or cells, the temperature and medium has to be suitably chosen (citation) (FIG. 19A).

[0833] We furthermore can show that our nanocellulose composite with all its components is a suitable object to be produced by 3D printing technology (scheme: FIG. 19A). A standard 3D printer can be used for this (Makerbot: Replicator 2; FIG. 19B; Pettis et al., 2012), however, as is known for 3D printing of biological objects such as tissues or cells, the temperature and medium has to be suitable chosen.

[0834] Objective or Main Aim:

[0835] The nanocellulose chip is demanding to produce using only molecular biology techniques, it is not easy to modify and the numbers produced are low. Furthermore, the biotechnological synthesis process differs clearly from typical production methods in computer industry (silicon-wafers) which are more convenient to handle, faster and easy to modify.

[0836] Solution:

[0837] We present here a 3D printer variant for the production of nanocellulose chips, which allows their efficient production in high numbers. This enhances the quality of the nanocellulose chips obtained, additives which serve to conserve and protect the smart card and the integrated DNA can be easily added. Moreover, specific smart molecules are particularly well suitable to serve as micro printers (smart actuators) and are easily integrated into the chip card by this approach.

Embodiment Example

[0838] Accordingly, this invention explains how these valuable nanocellulose chips are produced with the help of a 3D printer fast, convenient, flexible and at low cost (for a review of 3D printers see Scheufens M, 2014). For this a specific form of a 3D printer is used: a specific modification of the nanocellulose to become printable (printer matrix) and a specific type of additives in the printing matrix (proteins, DNA, fluorophores, nucleotides and chemicals; specific protein engineering constructs, as described herein). Together this achieves the final product of the improved nanocellulose chip in high quality and high numbers.

[0839] Specific Properties:

[0840] 3D printer (basic scheme in FIG. 19A): A basic version uses for printing the PolyJet printer (ink jet principle). It was invented by the Israel-based enterprise PolyJet that fused 2012 with Stratasys Ltd. This printer can use at the same time several inks, for instance plastics. It is particularly suited for our application, printing of biomolecules and nanocellulose.

[0841] There are already large-sized printers, for instance the Voxeljet till 421 meter size (voxeljet AG, Friedberg, Germany). We recommend for the invention in order to achieve smart chips from nanocellulose by 3D printing the following three printer types (3D): [0842] I. melting printing; [0843] II. photo polymerization: UV light polymerizes liquid, light-fragile substance. Patented by Chuck Hulls (1984), 3D systems which is world-wide market leader: EnvisionTEC. This allows high accuracy printing with layers of 15 micro meters), [0844] III. A 3rd approach is Laser sintering (this is expensive, but very good to use for fast and simple conservation of information stored in our smart cards made of nanocellulose).

[0845] Basic Matrix:

[0846] In particular suitable is pure nanocellulose as wells bacterial cellulose (BC)/polycaprolactone (PCL) nanocomposite films. For these the production with hot compression is known (Figueiredo et al., 2015) as well as composite films from poly(vinyl ethanol) and bifunctional coupled cellulose nano crystals (Sirvio et al., 2015) as well as polylactid latex/nanofibrillated cellulose bio-nanocomposites (Larsson et al., 2012).

[0847] Further additives contain pure DNA for information storage or as substrate. It can furthermore be used as adaptor DNA or oligo-macrame (Lv et al., 2015) or as pore-membrane designer (Langecker et al., 2012).

[0848] Specific constructs, suitable for our invention: PolyU CidI Polymerase (with BLUF-domain or light-gated control), PolyA CidI Polymerase (or similarly controlled), or specifically modified, as well as further (modified) polymerases, light-gated controlled for preference, as well as (similarly modified) exonucleases, furthermore (light-gated), GFP-constructs and other fluorescent proteins, as well as different DNA molecules (with modifications).

[0849] Optimize Printing:

[0850] The optimal application and concentration of the mixture and the optimal temperature are important.

Further Embodiments

[0851] Printing: An alternative to ink jet printing is the makerbot replicator printer (see FIG. 19 B; Pettis et al., 2012). Other 3D printer types may be adapted, such as the sinter printer (here additives have to be made heat stable) and a printer with melting and printed layers (Adrian Bowyer, University of Bath 2005). These can partly print themselves as they contain in their construction a large fraction of printed parts. [0852] The printed chips are an important interface to other computer chips (produced from semiconductor industry) and this as printed circuits; for interfacing to these chips our nanocellulose composite chips use light or electronic properties. [0853] The added biomolecules, in particular the actuators (preferentially light-gated), support printing as micro-printers and for micro patterning and for the molecular translation (DNA, RNA) of genes (or parts thereof) and further enhance the functionality of the printed nanocellulose composite towards an universal constructor, i.e. a high flexible nanomachine for the production and the printing of information processing circuits. [0854] Long term storage of DNA by incorporation of silica glass particles can be easily achieved by our 3D printing approach and the above listed 3D printers, an important difference to demanding chemistry proposed earlier (Glass et al., 2015).

[0855] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

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