Patent Application
20180298370
The present invention relates to a 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.
Nanocellulose is a term referring to nano-structured cellulose. This can be cellulose nanofibers (CNF) also called microfibrillated cellulose (MFC), nanocrystalline cellulose (NCC), or bacterial nanocellulose (BNC), which refers to nano-structured cellulose produced by bacteria. Nanocellulose/CNF or NCC can be prepared from any cellulose source material, but woodpulp is normally used.
At the moment, nanocellulose is produced in increasing amounts worldwide. For example, Kralisch et al., 2015 describe a molecular biological method for bacterial nanocellulose production, also used by the company JeNaCell GmbH (Jena, Germany). At Edinburgh University and Sappi Limited (Johannesburg, South Africa) use an energy efficient macrocopic process for converting wood biomass into nanocellulose. In the US, the company American Process Inc. also uses biomass for the production of nanocellulose. In Mumbai (India), the ICAR-CIRCOT pilot plant produces daily 10 kg of nanocellulose since October 2014. Furthermore, the association of nonwoven fabrics industry which names nanocellulose as “the amazing material that promises flexible displays, faster cars and bullet-proof suits” focusses on the use of algae and sun light for the production of nanocellulose. See e.g. the association's congress “Rise 2015” (from graphene and nanofibers to intelligent fabrics and wearable electronics—at INDA's Research, Innovation & Science for Engineered Fabrics Conference (RISE®) and Nanofibers for the Third Millennium (N3M), February 9-12, in Miami, Fla.).
Nanocellulose is used in a plurality of applications, such as disclosed in US 2015/0024379 A1, US 2014/0370179 A1, US 2014/0367059 A1, US 2014/0345823 A1, US 2014/0323714 A1, US 2014/0323633 A1, US 2014/0224151 A1, US 2014/0255688 A1, US 2014/0088223 A1, US 2014/0202517 A1.
Furthermore, nanocellulose complements and replaces other materials used so far as biomatrices for tissue replacements. Such materials are e.g. synthetic materials, such as polyisopropyl acrylamid which in combination with polyethylene glycol polymerizes in the body due to the body temperature to a stabile bioadhesice matrix (Vernengo et al., 2010). There are biopolymers of chitosan, collagen, alginate, gelatin, elastin, fibrin, hyaluronic acid or silk protein, which are applied as beads, sponges, molded paddings, hydrogel or primarily in liquid form (Allen et al., 2004, Meakin 2001, Wilke et al., 2004, Sebastine and Williams 2007, Gruber et at, 2006). Matrices of atelocollagen are suitable for the cultivation of human mesenchymal stem cells (hMSC) (Sakai et al., 2005; Sakai et al., 2006; Lee et al., 2012). Scaffolds made of a combination of chitosan and gelatin provide suitable conditions for the cultivation of intervertebral disk cells isolated from rabbits (Cheng et al., 2010). Alginate obtained from brown algae is a suitable matrix for the cultivation of intervertebral disk cells as well (Chou et al., 2009).
All this confirms, there is a need in the art for improved nanocellulose materials to become an intelligent material that can process or store information. There is a need in the art for improved nanocellulose material which is suitable or can be tailored for a plurality of uses.
According to the present invention this object is solved by a bacterial nanocellulose composite, said bacterial nanocellulose comprising apart from the nanocellulose matrix DNA or RNA or modified nucleotides or further components for information processing.
According to the present invention this object is solved by a bacterial nanocellulose composite, said bacterial nanocellulose comprising nanocellulose and
According to the present invention this object is solved by using the bacterial nanocellulose composite of the present invention
According to the present invention this object is solved by using the bacterial nanocellulose composite of the present invention
According to the present invention this object is solved by a printing, storage and/or processing medium comprising the bacterial nanocellulose composite of the present invention.
According to the present invention this object is solved by a smart card or a chip card comprising the bacterial nanocellulose composite of the present invention.
According to the present invention this object is solved by providing the bacterial nanocellulose composite of the present invention for use as a medicament.
According to the present invention this object is solved by providing the bacterial nanocellulose composite of the present invention for use in a method of treating wounds and/or for detecting wounds and wound healing and/or for monitoring wound healing.
According to the present invention this object is solved by providing the bacterial nanocellulose composite of the present invention for use in a method of tissue engineering.
According to the present invention this object is solved by a skin transplant, tissue implant or neuro transplant comprising the bacterial nanocellulose composite of the present invention.
According to the present invention this object is solved by providing the bacterial nanocellulose composite of the present invention for use in a method of stimulus conduction, muscle stimulation and/or for monitoring heartbeat.
According to the present invention this object is solved by electronic skin comprising the bacterial nanocellulose composite of the present invention.
According to the present invention this object is solved by a method for producing a nanocellulose composite chip, comprising the steps of
According to the present invention this object is solved by a nanocellulose composite chip obtained by the method of the present invention.
Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.
Bacterial Nanocellulose Composite
Summary/Abstract
The present invention provides a bacterial nanocellulose composite, said bacterial nanocellulose composite comprising DNA or RNA or modified nucleotides or further components for information processing.
Specifically these are
As discussed above, the present invention provides bacterial nanocellulose composite materials.
Said bacterial nanocellulose composite comprises nanocellulose and
The bacterial nanocellulose composite of the present invention can comprise one or more of each of the components (i) to (iii) (and optionally (iv) as well) and combinations of the components (i) to (iii), and optionally further component(s) (iv). The choice of the components will depend on the planned application of the bacterial nanocellulose composite, in particular molecular information processing.
The bacterial nanocellulose composite of the present invention can comprise at least one component (i);
for example one or more of component (i);
The term “bacterial nanocellulose” when used herein refers to nanocellulose made from bacteria, in particular with high grade, high purity and well controlled fibre size and structure. Plant made nanocellulose can only be used if it achieves similar high grade and properties as a nanocellulose composite.
The term “bacterial nanocellulose composite” when used herein refers to bacterial nanocellulose which comprises further components, as defined herein.
The bacterial nanocellulose composite of the present invention can comprise one or more of each of the components (i) to (iii) as well as combinations of the components (i) to (iii), and optionally further component(s). The choice of the components will depend on the planned application of the bacterial nanocellulose composite.
The term “sensor molecule” or “signal processing molecule” or “information processing molecule”—as interchangeably used herein—refers to a molecule or compound that senses a signal, such as light, temperature, ions, ligands, and/or electric current, and responds to the signal and/or processes the signal via a conformational change, an (enzymatic) reaction (such as DNA or RNA synthesis), translocation, and/or that transfers it to an actuator or effector.
The term “actuator” or “actuator molecule” or “effector molecule”—as interchangeably used herein—refers to a molecule or compound that further translates or processes or transmits the signal sensed and transferred from the sensor or signal processing molecule(s), such as via a conformational change, an (enzymatic) reaction (such as DNA or RNA synthesis), translation and protein expression.
In the application of the bacterial nanocellulose composite in chip technology, the “sensor molecule” or “signal processing molecule” is also referred to as “input”; and the “actuator molecule” or “effector molecule” is also referred to as “output”. The different substrates (further components (iv)) which are modified by both molecule types (further proteins, synthesis or degradation of nucleotides etc.) are referred to as “information processing” before the final output is created.
(i) Sensor or Signal Processing Molecules
The bacterial nanocellulose composite of the present invention can comprise at least one sensor or signal processing molecule.
As discussed above, the term “sensor molecule” or “signal processing molecule” or “information processing molecule”—as interchangeably used herein—refers to a molecule or compound that senses a signal, such as light, temperature, ions, ligands, and/or electric current, and responds to the signal and/or processes the signal via a conformational change, an (enzymatic) reaction (such as DNA or RNA synthesis), translocation, and/or that transfers it to an actuator or effector.
In the application of the bacterial nanocellulose composite in chip technology, the “sensor molecule” or “signal processing molecule” is also referred to as “input”.
Said sensor or signal processing molecule(s) is/are preferably light-inducible or light-responding sensor molecule(s), i.e. the signal is light.
The signal can also be temperature, ions, ligands, and/or electric current.
Said sensor or signal processing molecule(s) can be:
Said proteins (a) can comprise said light-inducible or light-responding sensor domain(s) either naturally, or said proteins (a) are fusions with said domains, preferably genetically engineered.
Said protein domain(s) can be enzymatically active domains or binding domains.
Furthermore, domain(s) of different proteins can be part of a construct with said light-inducible or light-responding sensor domain(s).
Preferably, the protein(s) comprising light-inducible or light-responding sensor domains are selected from:
In some embodiments, domain(s) of the above mentioned protein(s) are used, such as catalytic or enzymatically active domains and/or binding domains.
In one embodiment, the protein(s)/protein domain(s) comprising light-inducible or light-responding sensor domain(s) further comprise linker(s) and/or secretion signal(s) or signal peptide domain(s).
This e.g. allows for the protein(s) or protein domain(s) to locate to/to be transported, or the like, to certain positions within the fibres of the nanocellulose (composite).
The choice of the sensor molecule(s)/proteins will depend on the planned application of the bacterial nanocellulose composite.
For example: planned application as smart card/chip card or the like (DNA storage medium): Suitable proteins are nucleotide-specific polymerase constructs or other nucleotide processing and/or binding proteins/enzymes, such as DNA polymerase(s) and RNA polymerase(s), such as Cid1 polymerase, PolyU polymerase, μ DNA polymerase, terminal deoxyncleotidyl (TdT) polymerase, or active domains thereof.
Suitable sensor or signal processing molecule(s) are e.g.:
For example, for nucleotide-based information processing the sensor or signal processing molecule(s) (i.e. protein(s)) are
For Example: Planned Medical Application
For e.g. the application as an “intelligent” nanocellulose composite for medical applications (such as, intelligent plaster) suitable sensor or signal processing molecule(s) are embodied in the intelligent nanocellulose composite an can monitor the state of the wound, e.g. measure temperature, pH, inflammation (cytokines) and can also show by a change in fluorescence the resulting state.
Furthermore, the healing process should be improved by suitable programming the tissue or cells. For this the nanocellulose composite can contain as further component(s) growth promoting molecules such as growth factors (VEGF, EGF, PDGF), kinases, but also connective tissue stimulating components such as collagens. All these different components are well controlled, monitored and only selectively released in the nanocellulose composite including a suitable surface treatment of the nanocellulose (iii).
(ii) Actuator Molecules
The bacterial nanocellulose composite of the present invention can comprise at least one actuator or effector molecule.
As discussed above, the term “actuator” or “actuator molecule” or “effector molecule”—as interchangeably used herein—refers to a molecule or compound that further translates or processes or transmits the signal sensed and transferred from the sensor or signal processing molecule(s), such as via a conformational change, an (enzymatic) reaction (such as DNA or RNA synthesis), translation and protein expression.
In the application of the bacterial nanocellulose composite in chip technology, the “actuator molecule” or “effector molecule” is also referred to as “output”.
This embodiment is particularly suitable for uses in chip technology and as storage medium.
Preferably, the actuator or effector molecule(s) are enzymes or structure proteins so that an output or action is transmitted to the nanocellulose surface
Said actuator or effector molecule(s) can be light-inducible or light-responding molecule(s), i.e. the signal is light.
Said actuator or effector molecule(s) can be:
Said proteins (a) can comprise said light-inducible or light-responding sensor domain(s) either naturally, or said proteins (a) are fusions with said domains, preferably genetically engineered.
Preferably, the actuator or effector molecule(s) comprise light-inducible or light-responding domain(s)/protein(s) that respond to a light of different wavelength than the sensor or signal processing molecule(s).
In such embodiment, the molecules can be controlled individually from each other by the use of light of said different wavelengths.
Said protein domain(s) can be enzymatically active domains or binding domains.
Furthermore, domain(s) of different proteins can be part of a construct with said light-inducible or light-responding actuator or effector domain(s).
Preferably, the actuator or effector molecule(s)/protein(s) (optionally comprising light-inducible or light-responding sensor domains) are selected from:
In some embodiments, domain(s) of the above mentioned protein(s) are used, such as catalytic or enzymatically active domains and/or binding domains.
The choice of the actuator or effector molecule(s)/protein(s) will depend on the planned application of the bacterial nanocellulose composite.
In one embodiment, the protein(s)/protein domain(s) comprising light-inducible or light-responding domain(s) further comprise linker(s) and/or secretion signal(s) or signal peptide domain(s).
This e.g. allows for the protein(s) or protein domain(s) to locate to/to be transported, or the like, to certain positions within the fibres of the nanocellulose (composite).
Light Inducible or Light-Responding Domains
Preferably, the light-inducible or light-responding sensor molecule(s) or light-inducible or the light-responding sensor/actuator/effecor domain(s) comprise or are:
or
The BLUF domain (sensors of blue-light using FAD) is a FAD-binding protein domain. The BLUF domain is present in various proteins, primarily from bacteria, for example a BLUF domain is found at the N-terminus of the AppA protein from Rhodobacter sphaeroides. The BLUF domain is involved in sensing blue-light (and possibly redox) using FAD and is similar to the flavin-binding PAS domains and cryptochromes. The predicted secondary structure reveals that the BLUF domain has a novel FAD-binding fold.
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 protein—Euglena gracilis, Gomelsky and Klug, 2002; Slr1694—Synechocystis sp. Okajima et al., 2005) and regulation of transcription (e.g. AppA protein—Rhodobacter sphaeroides, Masuda and Bauer, 2005; Blrp—E. coli, Pesavento and Hengge, 2009). The proteins containing BLUF or similar domain was found also in Klebsiella pneumonia (Tyagi et al., 2013), Naegleria gruberi (Yasukawa et al., 2013), Acinetobacter baylyi (Bitrian et al., 2013) and many others organism. 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 transmit 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.
The BLUF domain can in particular be obtained as part of the YcgF gene and protein (Tschwori et al., 2009; Tschwori et al., 2012). DNA for the BLUF domain can, thus, in particular be gene ycgF (Accession number AAC74247.3) from E. coli.
See e.g. SEQ ID NO. 1, as listed in Database: UniProt/SWISS-PROT, Entry: BLUF_ECOLI
Light-oxygen-voltage-sensing (LOV) domains are protein sensors used by a large variety of higher plants, microalgae, fungi and bacteria to sense environmental conditions. In higher plants, they are used to control phototropism, chloroplast relocation, and stomatal opening, whereas in fungal organisms, they are used for adjusting the circadian temporal organization of the cells to the daily and seasonal periods. Common to all LOV proteins is the blue-light sensitive flavin chromophore, which in the signaling state is covalently linked to the protein core via an adjacent cysteine residue. LOV domains (Mart et al., 2016) are e.g. encountered in phototropins, which are blue-light-sensitive protein complexes regulating a great diversity of biological processes in higher plants (e.g. phototropin 2 in Arabidopsis thaliana, genbank accession CP002688.1) as well as in micro-algae.
Phototropins are composed of two LOV domains, each containing a non-covalently bound flavin mononucleotide (FMN) chromophore in its dark-state form, and a C-terminal Ser-Thr kinase. Upon blue-light absorption, a covalent bond between the FMN chromophore and an adjacent reactive cysteine residue of the apo-protein is formed in the LOV2 domain (Yao et al., 2008). This subsequently mediates the activation of the kinase, which induces a signal in the organism through phototropin autophosphorylation. In case of the fungus Neurospora crassa, the circadian clock is controlled by two light-sensitive domains, known as the white-collar-complex (WCC) and the LOV domain vivid (VVD-LOV). LOV domains have also been found to control gene expression through DNA binding and to be involved in redox-dependent regulation, like e.g. in the bacterium Rhodobacter sphaeroides.
Furthermore, the crystal structure of Lov1 Domain for instance of Phototropin2 from Arabidopsis thaliana (PDB code 2Z6D_B) is known in atomic detail (e.g. allowing an easier engineering, such as these for light-dependend control of the subsequent CidI polymerase, see below).
Amino Acid Sequence of Lov1 Domain:
Cryptochromes (CRYs) are a class of flavoproteins that are sensitive to blue light. They are found in plants and animals. Cryptochromes are involved in the circadian rhythms of plants and animals, and in the sensing of magnetic fields in a number of species.
The two genes Cry1 and Cry2 code for the two cryptochrome proteins CRY1 and CRY2. In insects and plants, CRY1 regulates the circadian clock in a light-dependent fashion, whereas, in mammals, CRY1 and CRY2 act as light-independent inhibitors of CLOCK-BMAL1 components of the circadian clock. In plants, blue light photoreception can be used to cue developmental signals.
Examples of fusion protein constructs of BLUF domains with polymerases or domains of polymerases are disclosed in German patent application of one of the inventors, DE 10 2013 004 584.3, which is enclosed herewith in its entirety.
Such examples are for instance:
Fluorescent Proteins and Protein Domains
In one embodiment, the sensor molecule(s) (i) and/or the actuator or effector molecule(s) (ii) comprise or are
In one embodiment, the fluorescent protein(s) or protein(s) comprising fluorescent domain(s) or fusion protein(s) with fluorescent protein(s) or domain(s) comprise
Embodiment where (i) and (ii) are Combined
In one embodiment, the sensor or signal processing molecule (i) and the actuator or effector molecule (ii) can be combined in one molecule or can be fused to each other.
For example,
(iii) Cells
The bacterial nanocellulose composite of the present invention can comprise cells.
This embodiment is particularly suitable for medical uses.
Examples for cells are skin cells, stem cells (such as mesenchymal stem cells).
For example, the bacterial nanocellulose composite can comprise mesenchymal stem cells when it is to be used in wound healing.
For example, the bacterial nanocellulose composite can comprise specific tissue cells when it is to be used in tissue engineering, such as artificial lung tissue cells (see e.g. Stratmann et al., 2014)
(iv) Further Components
The bacterial nanocellulose composite of the present invention can comprise further component(s).
Said further component(s) can be components for the sensor/actuator/effector molecule(s).
For example:
Said further component(s) can be further polymer(s).
For example:
Said further component(s) can be graphene or fullerene.
Graphene, for instance, serves better interfacing with electronic components.
Said further component(s) can also be marker(s), label(s).
For example: chromophores, fluorophores and/or radioisotopes.
They can, for instance, serve to enhance clarity of the output on the surface of the nanocellulose composite.
Said further component(s) can also be compounds supporting wound healing and/or stimulating (tissue) growth.
For example:
Said further component(s) can also be drugs, antibodies or antibody fragments.
The bacterial nanocellulose composite of the present invention can comprise combinations of said further component(s),
such as enzyme substrate(s) and cofactor(s) and ion(s),
such as (oligo)nucleotide(s) and further polymer(s),
and so on.
Nanocellulose Composite with Surface or Surface Layer
In one embodiment, the bacterial nanocellulose composite of the present invention forms or comprises a surface or surface layer.
Said surface or surface layer preferably comprises sensor or signal processing molecule(s) (i) which can be selected from:
For example, said sensor proteins or enzymes are active on the surface and/or active expressed at the surface, hence actuators for “printing”. One example are two component systems composed of sensors and actuators/responders as known from various bacteria; described in e.g. Krüger et al., 2012.
In one embodiment, said surface or surface layer optionally comprises further component(s), such as
These embodiments provide a nanocellulose composite with a surface suitable for electronic or optical properties to interface to electronic components or achieve output.
Thereby, the nanocellulose composite provides a natural surface. Modifying the surface by pores or modification of the nanocellulose itself yields electronic properties or provides optical properties. The nanocellulose composite for information processing can now use these optical and electronical properties for displaying the stored information (e.g. by fluorescence) or for interfacing electronically or optically with other electronic devices (e.g. smart phone, computer, glass-fibre cable).
Methods of Obtaining the Bacterial Nanocellulose and the Composite
Preferably, the bacterial nanocellulose is obtained via bacterial fermentation or bacterial expression.
For example, in
The bacterial nanocellulose can be obtained from plant sources and is then bacterially fermented.
For example, according to Kralisch et al. (2014) Komagataeibacter (previous name: Acetobacter or Gluconacetobacter) is used.
Growth medium: Hestrin-Schramm medium made from water, glucose, yeast extract plus pepton, pH buffering—wherein numerous alternative media, for instance from plants are known.
One advantage of the procedure according to Kralisch et al. is the obtainment of high quality bacterial nanocellulose on the surface of the culture with a continuous process for constant and efficient production of nanocellulose.
For example, according to Nobles and Brown (2008) cyanobacteria, in particular Synechococcus leopoliensis strain UTCC 100, are used.
Transfer of the nanocellulose synthesis into cyano bacteria can enhance the yield. Nanocellulose is generated in a bioreactor at moderate temperatures (25-30° C.) at the surface of the liquid culture (interface to air) as a structure stable hydro-polymer (solid phase fraction about 1%, hydrogel). The polymer is harvested at the surface.
From a molecular perspective, nanocellulose is generated between cell wall and external membrane of the bacterial cell by a cellulose synthase complex which produces nanocellulose as a quite long glucose chain molecule from UDP-glucose monomers. The glucose polymers leave the cell as cellulose elementary fibrils through pores at the surface and aggregate to microfibrils. This self-assembly together with cell division and branching resulting therefrom, leads to the characteristic three dimensional fiber network.
According to the present invention, the production of the bacterial nanocellulose (composite) relies on expression in E. coli. For details, see the examples. The described method allows an easy production as well as manipulation of the nanocellulose and the resulting nanocellulose composite.
There are different ways for “adding” or including or embedding the components (i) to (iv) to/into the bacterial nanocellulose:
In one embodiment, the sensor or signal processing molecule(s) (i) and/or actuator/effector molecule(s) (ii) and/or cell(s) (iii) and further component(s), if present, are embedded or encapsulated in the bacterial nanocellulose composite.
In this embodiment, the component(s) can be added to the bacterial nanocellulose.
The sensor or signal processing molecule(s) (i) and/or actuator/effector molecule(s) (ii) and/or further component(s) (iv), if present, can also be co-produced during the bacterial fermentation or bacterial expression of the bacterial nanocellulose itself.
Thereby, particular expression constructs and cell biological cell lines are utilized.
In one embodiment, the sensor molecule(s) (i) and/or actuator/effector molecule(s) (ii) and/or further component(s) (iv), if present, are covalently attached to the nanocellulose,
In this embodiment, the bacterial nanocellulose and/or the sensor/actuator/effector molecule(s)/further component(s) can comprise said linker, anchor groups.
The component(s) can be added to the bacterial nanocellulose or they can also be co-produced during the bacterial fermentation or bacterial expression of the bacterial nanocellulose itself. Thereby, particular expression constructs and cell biological cell lines are utilized.
In one embodiment, which comprises more than one of the components (i) to (iii) and optionally further component(s) (iv), one or more of said component(s) can be embedded or encapsulated whereas one or more of said components can be covalently attached.
The skilled artisan will be able to choose the most suitable way, dependent on the planned application/use of the bacterial nanocellulose composite.
Uses of the Bacterial Nanocellulose Composite
As discussed above, the present invention provides the use of the bacterial nanocellulose composite in material engineering and chip technology.
In particular, the present invention provides the use of the bacterial nanocellulose composite
As discussed above, the present invention provides the use of the bacterial nanocellulose composite in wound healing and tissue engineering.
In particular, the present invention provides the use of the bacterial nanocellulose composite
Preferably, the bacterial nanocellulose composite is used in form of a hydrogel, a foil, a layer, optical transparent paper.
Depending on the intended use, the composition of the nanocellulose composite changes, i.e. the components (i) to (iii) and optionally (iv) have to be chosen/combined.
For example:
(1) For Use in Information Storage and Processing: The nanocellulose composite of the present invention preferably comprises at least:
The synthesized nucleotides are for storage.
(2) For molecular information processing of RNAs and proteins:
The nanocellulose composite of the present invention preferably comprises at least:
(3) As output device and for connection to/interfacing with electronic components
The nanocellulose composite of the present invention preferably comprises at least:
as output device and for connection to electronic components (including typical refinement steps from chip manufacturing on the nanocellulose composite).
(4) For monitoring, e.g. wounds or wound healing
The nanocellulose composite of the present invention preferably comprises at least:
for monitoring (e.g. in wounds).
(5) For reprogramming wounds for optimal healing
The nanocellulose composite of the present invention preferably comprises at least:
(e.g. for intelligent plaster)
(6) As intelligent skin or tissue substitute
The nanocellulose composite of the present invention preferably comprises at least:
to become part of a tissue (e.g. for an artificial skin).
Uses in Material Engineering and Chip Technology
As discussed above, the present invention provides a printing, storage and/or processing medium comprising the bacterial nanocellulose composite of the present invention.
Said medium is preferably in form of a foil or a transparent display.
As discussed above, the present invention provides a smart card or a chip card comprising the bacterial nanocellulose composite of the present invention.
Said smart card or chip card optionally further comprises graphene and/or organic polymer(s).
Preferably, the bacterial nanocellulose composite is in the form of a hydrogel in the inside of the smart card or the chip card, preferably with a solid nanocellulose surface.
Medical Uses
As discussed above, the present invention provides the bacterial nanocellulose composite for use as a medicament.
As discussed above, the present invention provides the bacterial nanocellulose composite for use in a method of treating wounds.
As discussed above, the present invention provides the bacterial nanocellulose composite for use in detecting wounds and wound healing.
As discussed above, the present invention provides the bacterial nanocellulose composite for use in a method of monitoring wound healing.
In said method of treating wounds and/or for detecting wounds and wound healing and/or for monitoring wound healing, the bacterial nanocellulose composite preferably comprises
The bacterial nanocellulose composite is preferably a hydrogel.
As discussed above, the present invention provides the bacterial nanocellulose composite for use in a method of tissue engineering.
In said method, the bacterial nanocellulose composite preferably comprises
As discussed above, the present invention provides a skin transplant, tissue implant or neuro transplant comprising the bacterial nanocellulose composite of the present invention.
As discussed above, the present invention provides a tissue implant comprising the bacterial nanocellulose composite of the present invention.
As discussed above, the present invention provides a neuro transplant comprising the bacterial nanocellulose composite of the present invention.
Combined Uses
As discussed above, the present invention provides the bacterial nanocellulose composite for use in a method of stimulus conduction, muscle stimulation and/or for monitoring heartbeat.
In said method, the bacterial nanocellulose composite preferably comprises
preferably mesenchymal stem cells,
As discussed above, the present invention provides an electronic skin comprising the bacterial nanocellulose composite of the present invention.
Wound Healing and Tissue Engineering Methods
(1) The present invention provides a method of treating wounds.
Said method comprises the step of administering to a wound of a subject in need thereof a therapeutically active amount of the bacterial nanocellulose composite of the present invention.
(2) The present invention provides a method for detecting wounds and wound healing and/or for monitoring wound healing.
Said method comprises the step of administering to a wound of a subject in need thereof the bacterial nanocellulose composite of the present invention.
In above methods (1) and (2), the bacterial nanocellulose composite preferably comprises
The bacterial nanocellulose composite is preferably a hydrogel.
(3) The present invention provides a method of tissue engineering.
Said method can be an in vitro, ex vivo or in vivo method.
Said method (3) comprises the use of the bacterial nanocellulose composite of the present invention, which preferably comprises
(4) The present invention further provides a method of stimulus conduction, muscle stimulation and/or for monitoring heartbeat.
Said method (4) comprises the step of administering to a subject in need thereof the bacterial nanocellulose composite of the present invention.
The bacterial nanocellulose composite preferably comprises
3D Printing Method
As discussed above, the present invention provides a method for producing a nanocellulose composite chip.
Said method comprises the steps of
Preferably, the nanocellulose in step (1) is
Preferably, the 3D printer in step (2) is an ink jet printer, a sinter printer, a printer with melt layering.
As discussed above, the present invention provides nanocellulose composite chip obtained by said method.
Further Description of Preferred Embodiments
Our invention provides bacterial nanocellulose composite materials which contain DNA or RNA or modified nucleotides or further components for information processing. Moreover, our constructs (see detailed examples and explanations herein) as well as their broader principles allow the nanocellulose composite to become information processing (e.g. smart card, computer chip) as well as to become an intelligent material (e.g. to support wound healing).
In particular, the inventors have developed a nanocellulose composite comprising specific constructs and properties to work as a smart card/computer chip and/or to improve wound healing.
In particular, the present invention provides a bacterial nanocellulose composite, said bacterial nanocellulose comprising apart from the nanocellulose matrix DNA or RNA or modified nucleotides or further components for information processing.
The following versions are advantageous for all involved tasks:
Nanocellulose matrix (including suitable modified nanocellulose as well as modifying its surface)
with DNA or RNA or modified nucleotides and/or further components for information processing
(1) which is operated on by light-gated nucleotide-specific polymerase constructs or other nucleotide processing or nucleotide binding enzymes (e.g. Cid I polymerase, mu-polymerases, exonucleases, transcription factors, T4 polynulceotide kinase, adenyltransferase) including light-gated versions of these enzymes or fluorescent protein constructs (GFP, YFP, CFP protein fusions) including light-gated versions to achieve storage and information processing capabilities (smart card or computer chip). Nucleotides (DNA, RNA) are used as substrate and synthesized nucleotides for storage; i.e. the nucleotides represent the stored information (read-in, read-out);
OR
(2) with light-gated RNA polymerases, or protein translation system or enzymatic synthesis system or enzymes or sensors or light-gated versions of these enzymes to achieve molecular processing of information stored in nucleic acid or protein sequences; (“nano factory”);
OR
(3) pores (from proteins or nucleic acid) for electronic or optical properties or fluorescent proteins or transparent nanocellulose or nanocellulose with modifyable optical properties or organic polymers or graphene or fullerene or dyes or sensor proteins or enzymes (active on the surface, active expressed at the surface, hence actuators for “printing”) including typical refinement steps from classical computer chip technology to achieve interfacing with electronic components or representation of the results (output);
OR
(4) with sensor proteins and/or modified nanocellulose surface (including fluorescent proteins, monitoring proteins or light-gated versions of these or dyes) to monitor healing in wounds;
OR
(5) with growth factors, or kinases or receptors or enzymes or drugs or light-gated versions of these to reprogram wounds for optimal healing;
OR
(6) containing sensors or enzymes or pores, actuators or electronic parts to achieve an intelligent skin or tissue substitute.
The bacterial nanocellulose composite of the present invention can comprise one or more of each of the components (1) to (6) and combinations of the components (1) to (6), and optionally further component(s). The choice of the components will depend on the planned application of the bacterial nanocellulose composite, in particular molecular information processing.
These components are now further clarified:
Further explanations of the individual components:
Ad (1), (2) Said nucleotide processing (in (1)) or protein processing (in (2)) molecules are preferably light-gated processing molecules. This means they are fused to a light-sensitive protein domain such as the BLUF domain or LOV domain or a cryptochrome domain so that their information processing activity can be switched on or off by light according to the specific wave length sensed by the light-gating domain.
Ad (4) A “sensor molecule” as used herein refers to a molecule or compound that senses a signal, such as light, temperature, ions, ligands, electric current, and responds to the signal or processes the signal via a conformational change, an (enzymatic) reaction or translocation. Also this sensing can be switched ON or OFF by fusion to a light-gating domain.
Ad (1), (2) and (4) Preferably, these light-gating domain(s) (Conrad et al., 2014) comprise or are BLUF domain, LOV domain or a cryptochrome, as described above.
Ad (1) Preferably, the protein(s) for nucleotide-based information processing are comprised from
terminal deoxyncleotidyl (TdT) polymerase,
Ad (3) Preferably, the nanocellulose composite with a surface for electronic or optical properties to interface to electronic components or achieve output the modified surface (layer) is derived from:
In some embodiments, domain(s) of the above mentioned protein(s) are used, such as catalytic or enzymatically active domains and/or binding domains.
Ad (1) Examples of fusion protein constructs of BLUF domains with polymerases or domains of polymerases are disclosed in German patent application of one of the inventors, DE 10 2013 004 584.3, which is enclosed herewith in its entirety.
Such examples are for instance:
Ad (1, 2, 3, 4, 5): In one embodiment, the protein(s)/protein domain(s) comprising light-inducible or light-responding sensor domain(s) further comprise linker(s) and/or secretion signal(s) or signal peptide domain(s). This e.g. allows for the protein(s) or protein domain(s) to locate to/to be transported, or the like, to certain positions within the fibres of the nano cellulose (composite).
For example: planned application as chip card or smart card (DNA storage medium; i, ii, iii): Suitable proteins for nucleotide processing (i) are DNA polymerase(s) and RNA polymerase(s), such as Cid1 polymerase, PolyU polymerase, μ DNA polymerase, terminal deoxyncleotidyl (TdT) polymerase, or active domains thereof. Rapid readout is achieved by exonucleases, in particular with nucleotide specificity. Access of specific DNA strand-regions is achieved by DNA binding proteins, for example transcription factor binding proteins. Activity of any of these proteins can easily be monitored by fusing these proteins to a fluorescent protein domain e.g. GFP, YFP, CFP.
For controlling the activity of any of these proteins, light-gated protein domains are fused to these proteins. Resulting suitable light-gated information processing molecule(s) are thus:
Similarly, the protein sequence processing molecules (ii) as well as the nanocellulose surface properties (iii), e.g. pore proteins on the surface, can be controlled by light-gating them by fusion to a BLUF or other light-sensing domain and each can be monitored by fusion to a monitoring fluorescent domain. Again the nanocellulose composite is a huge advantage for compactly keeping and integrating all involved molecules together.
For the application as an intelligent nanocellulose composite for medical applications (intelligent plaster; iv, v, vi) suitable sensor molecule(s) embodied in the intelligent nanocellulose composite monitor the state of the wound, e.g. measure temperature, pH, inflammation (cytokinines) and show by a change in fluorescence the resulting state. Furthermore, the healing process should be improved by suitable programming the tissue or cells. For this the nanocellulose composite can contain growth promoting molecules such as growth factors (VEGF, EGF, PDGF), kinases, but also connective tissue stimulating components such as collagens. All these different components are well controlled, monitored and only selectively released in the nanocellulose composite including a suitable surface treatment of the nanocellulose (iii).
Ad (3) Actuator Molecules
The bacterial nanocellulose composite of the present invention comprises at least one information processing molecule in any of the embodiments (1 to 6). To deliver the output of the stored information by protein expression, by color change, or change of the nanocellulose surface properties in general, actuator molecules are used. The embodiment (3) is particularly suitable for getting a strong and easy readable output signal from the intelligent nanocellulose composite.
Said actuator molecules are preferably fluorescent molecule(s).
In a preferred embodiment, said actuator molecules are
In one embodiment (see e.g.
Strong colours for achieving a clear output signal from the nanocellulose composite are also Gaussia proteins and other fluorescent proteins.
Further possibilities include modifying the surface of the nanocellulose itself (in particular its transparency), insertion of pores (for interfacing with electronics and electronic read-out). The nanocellulose composite allows as an alternative also sandwich assays, use of dyes, of organic polymers or of graphenes to achieve a good output signal and interfacing ability with electronic components.
Ad (6) Cells
The bacterial nanocellulose composite of the present invention can comprise cells. This embodiment is particularly suitable for medical uses.
The basic form of the nanocellulose composite is here an intelligent plaster monitoring healing disturbance (pH change) by color change. Cells, however, turn the nanocellulose composite into a scaffold with cells for optimal integration into tissues. This in itself strongly augments the positive effects of the nanocellulose plaster. Furthermore, this can be exploited to more directly intensify the healing and regeneration process. Examples for cells to be used in the nanocellulose composite for this application are skin cells, stem cells (such as mesenchymal stem cells).
For example, the bacterial nanocellulose composite can comprise mesenchymal stem cells when it is to be used in wound healing.
For example, the bacterial nanocellulose composite can comprise specific tissue cells when it is to be used in tissue engineering (for instance it can use artificial lung tissue cells; see e.g. Stratmann et al., 2014)
The choice of the information processing molecule(s) and proteins in the nanocellulose composite will depend on the planned application of the bacterial nanocellulose composite.
Ad (7) Further components
The bacterial nanocellulose composite of the present invention can comprise further component(s).
These are preferably added if they can enhance the information processing capabilities of the composite either directly (smart card, computer chip) or the positive reprogramming of human body cells in medical applications.
In one embodiment, the nucleotide processing molecule(s) (1) and/or RNA/protein processing molecules (2) or surface modifying and output mediating actuator molecule(s) (3), sensor molecules (4), cellular reprogramming molecules (5) and/or cell(s) (6) and further component(s), if present, are embedded or encapsulated in the bacterial nanocellulose composite.
In this embodiment, the component(s) can be added to the bacterial nanocellulose.
The information processing molecule(s) (1) to (5) and/or further component(s) (7), if present, can also be co-produced during the bacterial fermentation or bacterial expression of the bacterial nanocellulose itself. Thereby, particular expression constructs and cell biological cell lines are utilized. This was tested and is most easily achieved for said molecules by expression from one construct or expression from several plasmids in one bacterial strain such as E. coli high expression strains.
In one embodiment, the information processing molecule(s) (1) to (6) and/or further component(s) (7), if present, are covalently attached to the nanocellulose,
This application claims priority of German patent applications DE 10 2015 005 307.8 and DE 10 2015 005 308.6 filed Apr. 27, 2015, the contents of which are hereby incorporated in their entirety by reference.
Embodiment as Smart Card or Storage Chip or Computing Chip
We start from an already established highly efficient genetic process for nanocellulose generation (Kralisch et al., 2015). Said process uses gram negative aerobic bacteria, for instance Komagataeibacter (earlier name: Acetobacter or Gluconacetobacter). Growth medium: Hestrin-Schramm medium made from water, glucose, yeast extract plus pepton, pH buffering—wherein numerous alternative media, for instance from plants are known. Furthermore we want to emphasize that also other bacteria can be used, in particular cyano bacteria, as described by the Brown group, University of Texas (Nobles and Brown, 2008). Transfer of the nanocellulose synthesis into cyano bacteria strongly enhances the yield. Nanocellulose is generated in a bioreactor at moderate temperatures (25-30° C.) at the surface of the liquid culture (interface to air) as a structure stable hydro-polymer (solid phase fraction about 1%, hydrogel). The polymer is harvested at the surface. From a molecular perspective, nanocellulose is generated between cell wall and external membrane of the bacterial cell by a cellulose synthase complex which produces nanocellulose as a quite long glucose chain molecule from UDP-glucose monomers. The glucose polymers leave the cell as cellulose elementary fibrils through pores at the surface and aggregate to microfibrils. This self-assembly together with cell division and branching resulting therefrom, leads to the characteristic three dimensional fiber network. In natural conditions the fiber network serves for protection against drying-out, enemies, lack of oxygen or nutrients as well as UV-radiation. These properties complement optimal other tissue implants (e.g. chondrofillerliquid).
Subsequently the nanocellulose is populated with sensors and actuators (selected proteins, which prepare the matrix for utilization as a chip;
An innovative smart card or even chip card is generated: A nanocellulose foil is armed with biological switches (proteins).
The following components are used for the improvement of chip cards from nanocellulose with organic switches:
There are already efforts for an optical transparent “paper” for electronic displays (Kralisch et al., 2014). Nanocellulose is already used as LED display in computer components since some time (Ferguson et al., 2012). However, there the nanocellulose is only used as transparent cover.
The essential novelty of our invention arises by the combination of the imbedded components with the nanocellulose. Thus there is the combination of a light-gated polymerase with nanocellulose. By this arises a chip card in which important substrates such as cofactors and nucleotides can be used in the chip card for many cycles, in particular for data storage with the help of the light gated polymerase (DPA 10 2013 004 584.3). Advantageous is also the combination of nanocellulose with biological storage molecules, in particular bacterial rhodopsines (Imhof et al., 2014; Yao et al., 2005; Barnhardt et al., 2004) to use the chip card like this for data storage.
b. Further Improvements
c. Embodiment as Intelligent Material for Broader Applications
Starting from the intelligent nanocellulose or nanocellulose foil it is possible to complement or modify the matrix polymer, in particular by usage of
For the main intended uses as tissue replacement or as smart card usually composites are produced (in particular according to “a)”, usage together with plastics as main component in chip cards, or according to “c)” usage of inert Silicon as tissue replacement). The further broader applications of the nanocellulose composite gain most of all from the advantages in the two main applications:
Intelligent Chip Card:
This can be the combination of nanocellulose with graphenes, or with organic polymers, including such which can serve as battery. Important is to state that we use nanocellulose hydrogel in the inside, since then the substrates etc. for the imbedded molecules described above are at hand. Starting from this, there is in particular the option to replace many components of metal nature (condensors, resistors, transistors) or from plastics with proteins or nanocellulose or polymers from a) to c) in this biologically transformed chip card.
Combined Embodiment:
Together, both approaches yield further synergies in the application of nanocellulose together with our specific embedded components, for instance for muscle stimulation, cardiac monitoring or similar medical applications or a competitor products to “electronic skin” (Tee et al., 2012, who, however, use instead of our above components metals, in particular nickel and self-healing plastics), in doing so, the skin transplants gets by these procedures much better sensor properties.
(1) Intelligent nanocellulose, in particular modified nanocellulose foil, obtained by including of specific signal processing molecules, cells or actuator molecules in the nanocellulose.
The intelligent nanocellulose is suitable as composites of cells and protein structures for the chip card technology.
(2) Intelligent nanocellulose of (1) characterized in that the nanocellulose is not only used as transparent material such as in the LED technique, but further more actively as chip card, since the nanocellulose obtains further advantageous information carrier features due to the embedded molecular-biological switches, namely specific sensor or actuator molecules, respectively.
(3) Use of the intelligent nanocellulose as “intelligent material”, in particular as detector/“intelligent dust” (such as in comparison to imprinting detection media etc).
(4) Use of the intelligent nanocellulose as printer, storage medium and processing medium.
(5) Use of the intelligent nanocellulose ecological/environmentally friendly computer chip or chip card with low content of plastics/synthetic materials and/or metals.
Embodiment Wound Healing
We start from an already established high efficient genetic process for nanocellulose generation (Kralisch et al., 2015). Said process uses gram negative aerobic bacteria, for instance Komagataeibacter (earlier name: Acetobacter or Gluconacetobacter). Growth medium: Hestrin-Schramm medium made from water, glucose, yeast extract plus pepton, pH buffering—wherein numerous alternative media, for instance from plants are known. Furthermore we want to emphasize that also other bacteria can be used, in particular cyano bakteria, as described by the Brown group, University of Texas (Nobles and Brown, 2008).
Transfer of the nanocellulose synthesis into cyano bacteria strongly enhances the yield. Nanocellulose is generated in a bioreactor at moderate temperatures (25-30° C.) at the surface of the liquid culture (interface to air) as a structure stable hydro-polymer (solid phase fraction about 1%, hydrogel). The polymer is harvested at the surface. From a molecular perspective, nanocellulose is generated between cell wall and external membrane of the bacterial cell by a cellulose synthase complex which produces nanocellulose as a quite long glucose chain molecule from UDP-glucose monomers. The glucose polymers leave the cell as cellulose elementary fibrils through pores at the surface and aggregate to microfibrils. This self-assembly together with cell division and branching resulting therefrom, leads to the characteristic three dimensional fiber network. In natural conditions the fiber network serves for protection against drying-out, enemies, lack of oxygen or nutrients as well as UV-radiation. These properties complement optimal other tissue implants (e.g. chondrofillerliquid).
Subsequently the nanocellulose is populated with sensors and actuators (selected proteins, which in particular show or support wound healing, respectively, or which prepare the matrix for utilization as a chip;
By this we obtain a nanocellulose (e.g. as hydrogel) populated with wound-healing promoting molecules and cells as novel tissue replacement.
a. Improved Tissue Replacement and Wound Transplant or Wound Cover:
The following proteins are particular useful for the usage as sensor and hence as monitors for wound healing: proteins for measuring, in particular from two component systems (or also with an aptamer-component), which then measure metabolites, temperature, ion concentrations, tension-compression (important in the implant) as well as interactions; furthermore the measurement read-out is transmitted by fluorescence (GFP component) or by gene expression change (two component systems) or other signals. Fluorescent proteins or two component systems are simply imbedded in the hydrogel and they glow to show their state.
Natural growth factors are active agents for wound healing (VEGF, Erythropoetin, NGF, EGF etc.) and can be used in our composite. The same applies to collagen I, II, X, aggrekan, catabolic matrix degrading enzyme MMP-2 as well as human mesenchymal stem cells which provide support as well. In this application the hydrogel is kept liquid and absorbable, such that it is typically completely absorbed after some time (typically within several weeks).
Specifically tested were different collagenes (active molecules, actuators) as well as GFP-constructs (sensors) to test the intactness of a nanocellulose implant, however, as described above, there are many further possibilities, for instance the integration of further sugar molecules (tissue sugar code) to the nanocellulose (glycomic), to strongly promote wound healing.
b. Further Embodiments
c. Further Embodiment as Intelligent Material for Broader Applications
Starting from an intelligent nanocellulose or nanocellulose foil it is possible to complement or modify the matrix polymer, in particular by usage of
For the main intended use as tissue replacement such composites are produced (in particular according to “c)” usage of inert Silicon as tissue replacement). The further broader application of the nanocellulose composite gains most of all from the advantages in the two main applications:
Wound Healing:
Particularly advantageous is the integration of “b)” and “c)” as printed circuit, this renders the surface again more sensitive and suitable to support wound healing while preventing problems in the healing process. These additions are novel in the combination with nanocellulose as matrix and promise decisive improvements. Furthermore, wound healing is simultaneously supported and monitored by proteins or sensors if suitably supplied.
Combined Embodiment:
Together, both approaches yield further synergies in the application of nanocellulose together with our specific embedded components, for instance for muscle stimulation, cardiac monitoring or similar medical applications or a competitor products to “electronic skin” (Tee et al., 2012, who, however, use instead of our above components metals, in particular nickel and self-healing plastics), in doing so, the skin transplants gets by these procedures much better sensor properties.
(1) Intelligent nanocellulose, in particular modified nanocellulose foil, obtained by including of specific signal processing molecules, cells or actuator molecules in the nanocellulose.
The intelligent nanocellulose is suitable as composites of cells and protein structures for wound healing (such as band-aid, transplant), characterized in that
(2) Use of the intelligent nanocellulose as intelligent skin transplant and for general monitoring of the health state.
(3) Use of the intelligent nanocellulose for stimulus conduction or for improved healing and as neuro transplant and for muscle stimulation, including the heart.
The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
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).
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.
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.
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.
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.
Output (bottom): light-gated exonuclease constructs (triangles) are fused to specific nucleotide-binding domains (squares) and trigger different fluorescent proteins for readout.
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 control—in 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.
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.
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
C, close up looks on the engineered biofilm (scales are indicated, focus: patterned region).
A, Control base-line level.
B, Active T4 kinase readout.
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 ⅓ of the fragment.
A, Comparing BLUF-PNK-GFP, BLUF-GFP, GFP construct, Fluorescence in the dark. All three show fluorescence, the additional BLUF-domain enhances fluorescence.
B, Comparing BLUF-GFP, control and BLUF-PNK-GFP construct. UV plus daylight shows that the BLUF-GFP constructs respond with fluorescence under daylight.
A, Verification of BLUF-coding sequence from the transfected bacteria (Rosetta strain) by PCR reaction.
B, Verification of BLUF-Cid1 (long) and BLUF-Cid1 (cut) from the transfected bacteria (M15 strain) by PCR reaction.
Results of a BLUF-GFP construct. No blue light leads to inactive BLUF domain and hence far less fluorescence.
Shown are cultured bacteria in Lysogeny broth under UV.
A, negative control, only non-transfected E. coli in LB media,
B, positive control, induced E. coli with GFP cultured in 20 ml of media,
C, induced E. coli with BLUF-GFP construct in 20 ml of media,
D, lysate of non-induced E. coli (negative control),
E, lysate of E. coli with BLUF-GFP construct.
Here we show light-gated (blue light mediate) control of GFP fluorescence.
The comparative study of different GFP expression in the BLUF-GFP construct under different conditions (magnification 100×).
A, 16 hrs of cultivation in daylight (phase contrast),
B, 16 hrs of cultivation in daylight (under UV),
C, 16 hrs of cultivation in dark (phase contrast),
D, 16 hrs of cultivation in dark (under UV),
E, 24 hrs of cultivation in daylight (phase contrast),
F, 24 hrs of cultivation in daylight (under UV),
G, 24 hrs of cultivation in dark (phase contrast),
H, 24 hrs cultivation in dark (under UV).
A, Shown is SDS-PAGE with protein lysates of recombinant BLUF and BLUF-Cid1 constructs.
Lane 1—marker, line 2—BLUF-GFP, lane 3—BLUF-Cid1 (cut), lane 4—BLUF-Cid1 (long), lane 5—BLUF-GFP, lane 6—negative control, lysate from the non-induced cells.
B, Western-blot analysis of different BLUF constructs. Spot A—BLUF-GFP, Spot 2—BLUF-Cid1 (cut), spot 3—BLUF-Cid1 (long).
Amplification of BcsA and BcsB. Line 1—BcsA, line 2—BcsB.
Visualization of E. coli transformed by BcsA/BcsB with fluorescent reporters.
A, BcsA protein fused with GFP,
B, BcsB protein fused with mCHERRY.
Here time-dependent expression of rBcsB.
Lane 1—whole 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.
Nanocellulose stained by mCHERRY protein.
A and C, stained nanocellulose under red fluorescence.
B and D, the corresponding picture with phase contrast.
E and F, negative control, only nanocellulose (non-stained) under UV (E) with corresponding phase contrast (F).
Nanocellulose stained by GFP protein.
A, stained nanocellulose under green fluorescence.
B, corresponding picture with phase contrast.
C and D, negative control, only nanocellulose (non-stained) under UV (C) with corresponding phase contrast (D).
Flexible 3D Printer used in the 3D printing experiments for the nanocellulose composite.
A, shows design and structural parameters of molecular beacons.
B, shows that molecular beacons in solution can have three phases: bound to target, closed and random coil.
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).
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).
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.
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.
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.
1. Materials and Methods
1.1 Structural and Statistical Predictions
Calculation of engineered mutations were performed using the SCA MATLAB toolbox published by Ranganathan et al. (see Halabi et al, 2009).
All tested primer constructs for different BLUF domains, specifically:
Construct series A—BLUF(cut)-Linker-Cid I
Construct series B—Cid I(A)-Linker-BLUF(cut)-Linker-Cid I(B)
Construct series C—Cid I-Linker-BLUF
In each series, different linkers were tested (see below).
1.2. Molecular Cloning and Tests
For cloning, the pGEM®-T easy Vector system (Promega Corp.) was used.
2. Results
2.1. Key Steps for Active DNA Storage
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.
2.2. Testing Principles of DNA Storage
See
3.3. Demonstrating Active DNA Storage Enzymes
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.
We summarize in the following all different combinations tested and the evidence collected for the construct activities:
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.
Furthermore, control experiments measured T4 kinase activity using fluorescent oligos compared to negative controls.
b) Three different BLUF-Cid I constructs test control of Cid I polymerase activity by BLUF.
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.
d) Direct monitoring within bacteria by GFP constructs. Constructs include: GFP alone, BLUF-GFP, BLUF-Cid I-GFP, BLUF-Cid I-BLUF-GFP.
Fluorescence is observed for the constructs and the BLUF domain controls Cid I as well as GFP activity.
The accession numbers for these different proteins and genes are as follows:
The used DNA for the Cid1 construct started with poly(A) polymerase Cid1 (accession number NP_594901) from the yeast Schizosaccharomyces pombe [972h-].
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.
As the sequences have been modified, the resulting nucleotide sequences are shown in the following.
2.4 Polynucleotide Kinase (PKN)
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.
2.5. BLUF/Cid I Construct
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.
Construct A—BLUF(cut)-Linker-Cid I
AAAAAA.GCGCGCGC.GGGCCC.AAGCTT.
ATGCTTACCACCCTTATT
TACGCGCCTGCT
GGTGGTGGTGGAAGCGGCGGCGGCGGCAGC
TOP
GGCGGAGGGAGC
AGCTACCAAAAGGTCCCT
CGCCAGAAAAAAACGGATGAACAATCTAACAAAAAATTGTTGAATGAAACCGATGGTGACAATTCTGAGTGA
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.
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.
MNISSAQFIPGVHIVEEIEAEIHKNLHISKSCSYQKVPNSHKEFTKFCYEVYNEIKISDKEFKEKRAALDTLRLC
AAAAAA.GCCCTT.GGGCCC.AAGCTT.
ATGAACATTTCTTCTGCA
ATGAACATTTCTTCTGCACAATTTATTCCTGGTGTTCACACAGTTGAAGAGATTGAGGCAGAAATTCACAAAAAT
TTACATATTTCAAAAAGTTGTAGCTACCAAAAGGTCCCTAATTCGCACAAGGAATTTACGAAGTTTTGCTATGAA
CCCTCCGCCGCCAGGATCTTCAATCGCAAG
ATTGAAGATCCT
GGCGGCGGAGGGAGTGGTGGCGGAGGGTCA
GGCGGCGGCAGC
ATGCTTACCACCCTTATT
TACGCGCCTGCT
GGAGGAGGAGGATCCGGGGGAGGCGGTTCT
STOP
GGCGGGGGCAGC
TTCGAGATTTCACATAAT
CGTCGCCAGAAAAAAACGGATGAACAATCTAACAAAAAATTGTTGAATGAAACCGATGGTGACAATTCTGAGTGA
2.7. Cid I/BLUF (Complete) Construct
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.
Construct C—Cid I-Linker-BLUF
MNISSAQFIPGVHTVEEIEAEIHKNLHISKSCSYQKVPNSHKEFTKFCYEVYNEIKISDKEFKEKRAALDTLRLC
AAAAAA.GGGCCC.AAGCTT.
ATGAACATTTCTTCTGCA
ATGAACATTTCTTCTGCACAATTTATTCCTGGTGTTCACACAGTTGAAGAGATTGAGGCAGAAATTCACAAAAAT
TTACATATTTCAAAAAGTTGTAGCTACCAAAAGGTCCCTAATTCGCACAAGGAATTTACGAAGTTTTGCTATGAA
ACCTCCTCCTCCGGCCTCCTCAAATAATGA
TTTGAGGAGGCC
GGAGGAGGAGGTAGCGGTGGCGGAGGGTCA
GGCGGCGGGAGT
ATGCTTACCACCCTTATT
2.8. BLUF/Cid I/GFP (Preparation) Construct
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.
Construct D—BLUF(Cut)-Linker 1-Cid I-Linker 2-BLUF(Cut Long)-GFP(Prepare)
AAAAAA.CGCGCGCGC.CTGCAG.AGATCT.
ATGCTTACCACCCTTATT
TACGCGCCTGCT
GGTGGTGGTGGTTCTGGTGGTGGTGGTAGT
TOP
GGAGGAGGGAGC
AGCTACCAAAAGGTCCCT
CGCCAGAAAAAAACGGATGAACAATCTAACAAAAAATTGTTGAATGAAACCGATGGTGACAATTCTGAGTGA
ACCTCCTCCGCCTCACTCAGAATTGTCACC
AATTCTGAGTGA
GGCGGAGGAGGTAGCGGTGGCGGAGGGTCA
GGTGGGGGAAGT
ATGCTTACCACCCTTATT
2.9. BLUF/GFP (Preparation) Construct
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.
The predicted change in GFP activity level is shown in
Construct E—BLUE (Cut)-GFP (Prepare)
AAAAAA.CTGCAG.AGATCT.
ATGCTTACCACCCTTATTTATCGTAGC
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 protein—Euglena gracilis, Slr1694—Synechocystis sp.) and regulation of transcription (e.g. AppA protein ˜Rhodobacter sphaeroides, Blrp—E. 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.
Main Aim:
To produce BLUF and BLUF-Cid1 in E. coli expression system. See
Tools:
A circular DNA plasmid pPK-CMV-F1 vector with inserted BLUF domain with GFP on C-terminus (BLUF-GFP construct, see
Used DNA for the BLUF: gene ycgF (accession number AAC74247.3, see SEQ ID NO. 1.) from the E. coli strain DH5-α,
amplicon from 1-375 nt (125 AA), see SEQ ID NOs. 6 and 7.
Used DNA for the Cid1: poly(A) polymerase Cid1 (accession number NP_594901) from the Schizosaccharomyces pombe [972h-]
SEQ ID NO. 9.
Used Primers:
Constructs BLUF-GFP:
BLUF-GFP FW:
(including restriction sites for PstI and BglII)
BLUF-GFP RV:
(including restriction sites for XhoI and HindIII)
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-α and E. coli strain Rosetta (chemical transformation) were used.
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.
After transformation, bacteria with BLUF-GFP construct were cultured in Lysogeny broth (LB) and the protein was expressed using 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (
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 (
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 (3×10 sec cycles). The cell debris was removed by centrifugation and the supernatant contains proteins were separated by PAGE under reducing conditions. As seen in
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 (
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 (
Detailed functional proof of the observed correct fluorescence activity of the constructs requires polymerase or kinase activity monitoring using a fluorescent oligonucleotide (
In addition, this was achieved with different Cid I polymerase constructs,
as well as direct monitoring of fluorescence in read-out BLUF-GFP constructs
1. Molecular Beacon Assay
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.
We then generated the following beacons and oligonucleotides/primers with fluorophor TAMRA and the quencher BHQ2 for our experiments:
3.1 Control Experiments and Positive Controls Using DNA as Well as Klenow Fragment:
Beacon MB_1 (DNA):
This required the corresponding “Oligo_A” (DNA):
For the experiments with the Klenow-fragment, the primer “Oligo_B” (DNA) was used:
3.2 for the Experiments with the Cid1-Poly-U-Polymerase we Used the Following Oligonucleotides:
Beacon MB_2_Poly-U (DNA):
With the corresponding oligonucleotide to open the beacon (positive control) “Oligo_PUr” (RNA):
For the activity monitoring of the Cid1-Polymerase, the primer “Oligo_PriUr” (RNA) was used:
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
2. Light Microscopy Test
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 (
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.
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.
Nanocellulose can be used in various fields of industry; pharmaceutical, food production, textile, electronic, cosmetic and many more areas.
The recombinant DNA technology is routinely used in agriculture, food industry and medicine, but currently there is a new challenge—to 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.
Main Aim:
To produce nanocellulose in E. coli expression system.
Tools:
A circular DNA plasmid pQE-30-mCHERRY-GFP vector with inserted BcsA/BcsB unit, see
Used DNA for BcsA: gene bcsA—Cellulose synthase catalytic subunit [UDP-forming] (accession number AAB18510.1.) from the E. coli strain DH5-11, amplicon from 34-2610 nt (858 AA).
See SEQ ID NO. 32 for the full length amino acid sequence (872 aa), as shown in Database: UniProt/SWISS-PROT, Entry: BCSA_ECOLI.
Used DNA for the BcsB: gene bcsB—Cyclic di-GMP-binding protein (accession number AAB18509.1.) from the E. coli strain DH5-α, 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.
Used Primers:
BcsA-GFP Construct:
BcsA F:
(including restriction site for BamHI)
BcsA R:
(including restriction site for SalI)
BcsB-mCHERRY Construct
BcsB F:
(including restriction site for KpnI)
BcsB R:
(including restriction site for BlpI)
After amplification of BcsA and BcsB coding sequences (
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 (
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-GFP—130 kDa, GFP—30 kDa), but the BcsB was significantly overexpressed (predicted MW for the BcsB is 86 kDa, BcsB-mCHERRY—112 kDa, mCHERRY 26 kDa) (
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×,
We furthermore can show that our nanocellulose composite with all its components is a suitable object to be produced by 3D printing technology.
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) (
We furthermore can show that our nanocellulose composite with all its components is a suitable object to be produced by 3D printing technology (scheme:
Objective or Main Aim:
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.
Solution:
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.
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.
Specific Properties:
3D printer (basic scheme in
There are already large-sized printers, for instance the Voxeljet till 4×2×1 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):
Basic Matrix:
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).
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).
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).
Optimize Printing:
The optimal application and concentration of the mixture and the optimal temperature are important.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102015005307.8 | Apr 2015 | DE | national |
| 102015005308.6 | Apr 2015 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/059436 | 4/27/2016 | WO | 00 |
