Hierarchical live body DNA data storage system and method of operating the same

Abstract

The application discloses a hierarchical living body DNA data storage system and a running method thereof. The system comprises a database array, the database array is composed of a plurality of storage discs, and a plurality of living body DNA information storage units are arranged on the storage discs. The living body DNA information storage unit comprises a microsphere encapsulation matrix, and the microsphere encapsulation matrix encapsulates an engineering bacterium. The engineering bacterium carries an information plasmid, and the information plasmid comprises a phenotype index unit and an information sequence unit. The information sequence unit is used for storing a DNA coding sequence corresponding to digital information to be stored. The phenotype index unit is used for generating a detectable phenotype signal, and the detectable phenotype signal is used as a retrieval index of the DNA coding sequence. The hierarchical storage structure is constructed, and the detectable phenotype signal is used as the retrieval index of the DNA coding sequence, so that the ordered organization storage of the living body DNA information and the identifiable retrieval of a target storage unit are realized.

Description

Technical Field

[0001] This invention relates to the field of DNA information storage technology, specifically to a hierarchical living DNA data storage system and its operation method. Background Technology

[0002] As data volumes continue to grow, traditional silicon-based storage media are gradually approaching their physical limits in terms of storage density, energy consumption, and long-term preservation capabilities, prompting researchers to explore novel information storage media. DNA molecules, with their high information storage density and good chemical stability, have become an important research direction for novel data storage media. To improve the preservation stability of DNA information, current technologies typically encapsulate DNA in polymers, hydrogels, or nanomaterials for protection.

[0003] Existing DNA storage technologies mainly fall into two categories: in vitro storage and in vivo storage. In vitro storage typically preserves DNA molecules directly as information carriers; in vivo storage utilizes cell plasmids or genomes as information carriers, allowing information to exist in the form of genetic material within the organism. Compared to in vitro storage, in vivo storage can maintain the continuity of information to a certain extent through cell replication and repair mechanisms, thus possessing potential for sustainable storage.

[0004] However, current technologies focus more on molecular-level design, such as DNA encoding, molecular encapsulation, and gene editing, with relatively insufficient research on the overall organizational structure of living DNA storage systems. Especially in the context of living DNA storage, there is a lack of comprehensive system solutions for how to organize multiple storage units in an orderly manner, how to form higher-level storage structures, and how to establish retrieval indexes corresponding to the stored information. Existing living DNA information storage units are typically distributed, lacking the hierarchical organizational structure of traditional storage systems, which is detrimental to the systematic storage and management of large-scale living DNA information.

[0005] Furthermore, in living DNA storage, information is usually stored in plasmids or genomes inside cells. If there is a lack of detectable indexes corresponding to the stored information, it is difficult to effectively distinguish the target storage unit and locate it for subsequent retrieval.

[0006] Therefore, how to construct a live DNA data storage system with a hierarchical organizational structure and detectable indexes in the storage units has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] In order to solve at least one of the technical problems in the background art, the present invention proposes a hierarchical living DNA data storage system and its operation method.

[0008] In one aspect, the present invention provides a hierarchical live DNA data storage system, the system comprising a database array, the database array being composed of multiple storage disks, and multiple live DNA information storage units being disposed on the storage disks; The live DNA information storage unit includes a microsphere encapsulation matrix, which encapsulates engineered bacteria; the engineered bacteria carry an information plasmid, which includes a phenotypic index unit and an information sequence unit; the information sequence unit stores the DNA coding sequence corresponding to the digital information to be stored; the phenotypic index unit can generate a detectable phenotypic signal during detection, and the detectable phenotypic signal is used as a retrieval index for the DNA coding sequence.

[0009] Optionally, the hierarchical live DNA data storage system further includes: a retrieval module, used to determine the target storage disk corresponding to the digital information to be retrieved based on preset data index information, and to determine the target live DNA information storage unit corresponding to the digital information to be retrieved by matching the detectable phenotypic signal of each live DNA information storage unit in the target storage disk with the target detectable phenotypic signal corresponding to the digital information to be retrieved.

[0010] Optionally, the hierarchical live DNA data storage system further includes: An engineered bacteria release module is used to release engineered bacteria from the target live DNA information storage unit; The information reading module is used to extract information plasmids from the released engineered bacteria, sequence and decode the extracted information plasmids to obtain the digital information to be retrieved.

[0011] Optionally, the hierarchical live DNA data storage system further includes: The biological processing module is used to culture and amplify the released engineered bacteria to obtain amplified engineered bacteria; The repackaging module is used to mix the amplified engineered bacteria with the packaging matrix and repackage them into a new live DNA information storage unit, and then return the new live DNA information storage unit to the target storage disk.

[0012] Optionally, the biological processing module is further used to rewrite the DNA coding sequence on the information plasmid in the released engineered bacteria in situ, and to culture and amplify the engineered bacteria after the in situ rewriting to obtain the rewritten and amplified engineered bacteria. The repackaging module is also used to mix the rewritten and amplified engineered bacteria with the packaging matrix and repackage them into a new live DNA information storage unit, and place the new live DNA information storage unit into a predetermined storage disk.

[0013] Optionally, the engineered bacteria also carry an editing helper plasmid, which can target and cut old information sites on the information plasmid and write new information fragments in situ through homologous recombination, thereby realizing the in situ rewriting of the information stored on the information plasmid. The biological processing module is specifically used to rewrite the DNA coding sequence on the information plasmid in the released engineered bacteria in situ, based on the editing helper plasmid in the released engineered bacteria.

[0014] Optionally, the engineered bacteria release module releases the engineered bacteria in the target living DNA information storage unit through solvent dissolution or physical triggering.

[0015] Optionally, the hierarchical live DNA data storage system further includes: The freeze-drying module is used to freeze-dry the live DNA information storage unit in the database array so that the live DNA information storage unit is preserved in a freeze-dried state. The rehydration module is used to rehydrate the freeze-dried live DNA information storage unit, wherein the freeze-dried live DNA information storage unit can still be identified and selectively extracted based on the detectable phenotypic signal after rehydration.

[0016] Optionally, the detectable phenotypic signal is a phenotypic signal that can be identified by flow cytometry detection and / or flow cytometry sorting; the phenotypic indexing unit is used to regulate fluorescent protein expression to form the detectable phenotypic signal.

[0017] Optionally, the engineered bacteria also carry an editing helper plasmid, which can target and cut old information sites on the information plasmid and write new information fragments in situ through homologous recombination, thereby realizing in situ rewriting of the information stored on the information plasmid; the information plasmid and the editing helper plasmid constitute a mutually orthogonal dual plasmid system.

[0018] Optionally, the live DNA information storage unit is a bacterial microsphere, which is formed from a cross-linkable matrix material. The bacterial microsphere is formed from droplets carrying the engineered bacteria in a droplet microfluidic two-phase system, which are then solidified or cross-linked.

[0019] In another aspect, the present invention provides a method for operating a hierarchical live DNA data storage system, the method being applied to the aforementioned hierarchical live DNA data storage system, the method comprising: Upon receiving an information retrieval instruction, the target storage disk corresponding to the digital information to be retrieved is first determined based on the preset data index information. Then, the target live DNA information storage unit corresponding to the digital information to be retrieved is determined by matching the detectable phenotypic signal of each live DNA information storage unit in the target storage disk with the target detectable phenotypic signal corresponding to the digital information to be retrieved. Release the engineered bacteria from the target living DNA information storage unit; Information plasmids are extracted from the released engineered bacteria, and the extracted information plasmids are sequenced and decoded to obtain the digital information to be retrieved.

[0020] Optionally, the operation method of the hierarchical live DNA data storage system further includes: The released engineered bacteria were cultured and amplified to obtain the amplified engineered bacteria; The amplified engineered bacteria are mixed with the encapsulation matrix and repackaged into a new live DNA information storage unit, which is then returned to the target storage disk.

[0021] Optionally, the operation method of the hierarchical live DNA data storage system further includes: The block rewrites the DNA coding sequence on the information plasmid in the released engineered bacteria in situ, and then culturees and amplifies the engineered bacteria that have completed the in situ rewriting to obtain the rewritten and amplified engineered bacteria. The rewritten and amplified engineered bacteria are mixed with the encapsulation matrix and repackaged into a new live DNA information storage unit, which is then placed into a predetermined storage disk.

[0022] The beneficial effects of this invention are as follows: This invention establishes a hierarchical organizational structure for storing live DNA information by placing multiple live DNA information storage units on storage disks and further forming a database array from these disks. This avoids the inconvenience of managing existing live DNA information storage units that are scattered. Furthermore, within each live DNA information storage unit, the information sequence unit in the information plasmid stores the DNA coding sequence corresponding to the digital information to be stored, and the phenotypic index unit generates a detectable phenotypic signal as a retrieval index for the DNA coding sequence. This allows the storage unit to possess an identifiable and distinguishable index while carrying information, facilitating subsequent retrieval and location of the target live DNA information storage unit. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a schematic diagram illustrating the construction, usage process, and functions of a live DNA information storage unit; Figure 2 This is a schematic diagram of the system architecture and hierarchical retrieval structure; Figure 3 It is a flowchart of the automated operation of the storage system; Figure 4 This is a schematic diagram of the system architecture; Figure 5 This is a flowchart of the operation method of a hierarchical live DNA data storage system. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] Those skilled in the art will understand that embodiments of the present invention can be provided as methods, systems, or computer program products. Therefore, the present invention can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the present invention can take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0026] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification, claims and accompanying drawings of this invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such processes, methods, products or devices.

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] like Figure 4 As shown, in one embodiment of the present invention, the present invention provides a hierarchical live DNA data storage system, including a database array, the database array being composed of multiple storage disks, and multiple live DNA information storage units being disposed on the storage disks.

[0029] In this embodiment of the invention, the database array serves as the upper-level storage organization structure of the entire live DNA data storage system, centrally configuring and orderly managing multiple storage disks. The multiple storage disks can be arranged in a predetermined manner within the same array frame to form a data storage architecture with a hierarchical organizational relationship. This allows the originally dispersed live DNA information storage units to be categorized, carried, and centrally organized, thereby providing a structural foundation for subsequent data retrieval, reading, and updating operations.

[0030] In this embodiment of the invention, the storage disk is used to carry multiple live DNA information storage units. The storage disk can be a sheet-like carrier, a disk-like carrier, or other carrier structure capable of arranging multiple live DNA information storage units in an array. Each live DNA information storage unit can be spaced apart on the storage disk to facilitate differentiation, management, and retrieval of different live DNA information storage units. After multiple storage disks are further combined into a database array, a hierarchical organizational structure of live DNA information storage units—storage disks—database array can be formed at the system level, thereby improving the organized storage capability of live DNA information.

[0031] In this embodiment of the invention, the live DNA information storage unit includes a microsphere encapsulation matrix, in which engineered bacteria are encapsulated. The microsphere encapsulation matrix forms a physical embedding and protective structure for the engineered bacteria, allowing them to exist on the storage disk in a microsphere-like, unitized form. By employing microsphere encapsulation, the engineered bacteria can be organized and arranged in discrete units, and the live DNA information storage unit can be independently identified and accessed as the smallest information storage entity.

[0032] In this embodiment of the invention, the engineered bacteria carry an information plasmid. The information plasmid includes a phenotypic index unit and an information sequence unit. The information sequence unit stores the DNA coding sequence corresponding to the digital information to be stored. That is, the digital information to be stored is pre-encoded to form a corresponding DNA coding sequence, which is then written into the information sequence unit, so that the digital information can be stored within the engineered bacteria in the form of a DNA sequence. Thus, the living DNA information storage unit is not only a biological carrier unit but also a data unit that actually carries DNA-coded information.

[0033] In this embodiment of the invention, the phenotypic indexing unit is used to generate a detectable phenotypic signal, which serves as a retrieval index for the DNA coding sequence. That is, in the engineered bacteria, in addition to storing the DNA coding sequence corresponding to the digital information through the information sequence unit, the phenotypic indexing unit also assigns a detectable phenotypic identifier to the living DNA information storage unit. Subsequently, when it is necessary to retrieve target information, the detectable phenotypic signal can be used to first identify and locate the living DNA information storage unit, and then the DNA coding sequence stored within it can be read. In this way, the living DNA information storage unit can possess indexable and identifiable characteristics while carrying information.

[0034] Furthermore, a clear hierarchical relationship is formed between the database array, storage disks, and live DNA information storage units in this embodiment. The live DNA information storage unit, as the smallest information storage unit, is used to actually carry the DNA coding sequence corresponding to the digital information to be stored; the storage disk, as an intermediate carrying layer, is used to centrally house multiple live DNA information storage units; and the database array, as the upper-level organizational structure, is used to uniformly manage multiple storage disks. Through this arrangement, live DNA information storage can be further expanded from unit-level storage to disk-level organization and array-level management, thereby facilitating the systematic storage of large-scale live DNA information.

[0035] Optionally, the multiple storage disks can be configured to correspond to different data categories, different data batches, and / or different data partitions; the multiple live DNA information storage units on each storage disk can each correspond to different DNA coding sequences and their corresponding detectable phenotypic signals. This further enhances the data organization and scalability of the entire hierarchical live DNA data storage system.

[0036] Based on the above technical solution, in this embodiment of the invention, multiple storage disks are set in the database array, and multiple live DNA information storage units are set on each storage disk. At the same time, the DNA coding sequence is stored in the live DNA information storage unit using information sequence units, and detectable phenotypic signals are generated as retrieval indexes using phenotypic index units. This realizes hierarchical organization and indexable expression of live DNA information, providing a foundation for the identification, location and retrieval of target live DNA information storage units.

[0037] like Figure 4 As shown, in one embodiment of the present invention, the hierarchical live DNA data storage system further includes a retrieval module. The retrieval module is used to determine the target storage disk corresponding to the digital information to be retrieved based on preset data index information, and to determine the target live DNA information storage unit corresponding to the digital information to be retrieved by matching the detectable phenotypic signals of each live DNA information storage unit in the target storage disk with the target detectable phenotypic signals corresponding to the digital information to be retrieved.

[0038] In this embodiment of the invention, the retrieval module is used to implement hierarchical retrieval of target live DNA information storage units. This hierarchical retrieval is mainly reflected in two levels: first, at the disk level of the database array, the target storage disk corresponding to the digital information to be retrieved is determined; second, within the target storage disk, each live DNA information storage unit is further identified and matched to determine the actual target live DNA information storage unit storing the target information. This disk-level and unit-level retrieval method avoids directly screening all live DNA information storage units one by one across the entire database array, thereby improving the targeting and effectiveness of the retrieval.

[0039] In this embodiment of the invention, the preset data index information is used to characterize the correspondence between the digital information to be retrieved and the target storage disk. That is, when the system pre-writes data, disk-level index information corresponding to each piece of digital information can be established simultaneously. Subsequently, when a retrieval request for a specific piece of digital information is received, the retrieval module can first determine the corresponding target storage disk from the database array based on the preset data index information. This allows the retrieval scope to be limited to the target storage disk, rather than indiscriminately searching the entire database array.

[0040] In this embodiment of the invention, after identifying the target storage disk, the retrieval module further identifies each live DNA information storage unit in the target storage disk. Specifically, the retrieval module obtains the detectable phenotypic signal corresponding to each live DNA information storage unit in the target storage disk and matches it with the target detectable phenotypic signal corresponding to the digital information to be retrieved. If the detectable phenotypic signal of a certain live DNA information storage unit matches the target detectable phenotypic signal, then it can be determined that the live DNA information storage unit is the target live DNA information storage unit storing the DNA coding sequence corresponding to the digital information to be retrieved.

[0041] It is understood that, in this embodiment, the target detectable phenotypic signal can be determined by the index relationship corresponding to the digital information to be retrieved. That is, when the system completes the writing of digital information, a correspondence between the digital information to be stored, the DNA coding sequence, and the detectable phenotypic signal can be established simultaneously; when performing information retrieval, the target detectable phenotypic signal corresponding to the digital information to be retrieved can be obtained, and the corresponding live DNA information storage unit can be further located within the target storage disk using the target detectable phenotypic signal.

[0042] Furthermore, after the retrieval module identifies the target live DNA information storage unit within the target storage disk, it can output this target live DNA information storage unit to the subsequent processing unit for operations such as engineered bacteria release, information plasmid extraction, and sequencing decoding. In other words, the retrieval module in this embodiment primarily functions to locate the target storage disk from the database array and, within the target storage disk, the target live DNA information storage unit, thus providing a prerequisite for subsequent information retrieval.

[0043] In this embodiment of the invention, the retrieval module enables the live DNA data storage system to possess not only hierarchical storage capabilities but also hierarchical retrieval capabilities that match the hierarchical storage structure. The retrieval module first determines the target storage disk based on preset data index information, and then determines the target live DNA information storage unit based on the matching relationship between the target detectable phenotypic signal and the detectable phenotypic signal of the live DNA information storage unit, thereby achieving a layer-by-layer retrieval from the disk level to the unit level. This improves the accuracy and orderliness of the target information retrieval process.

[0044] In some implementations, the preset data index information can be pre-stored in a control unit, index database, or other data management module. Upon receiving a retrieval request corresponding to the digital information to be retrieved, the retrieval module can call the preset data index information to determine the corresponding target storage disk. Further, within the target storage disk, the retrieval module can determine the target live DNA information storage unit by detecting, identifying, and matching detectable phenotypic signals. The specific implementation methods of the detectable phenotypic signals, such as flow cytometry detection, flow cytometry sorting, and fluorescence signal recognition, will be further explained in subsequent embodiments.

[0045] Based on the above technical solution, the retrieval module in this embodiment first determines the target storage disk and then determines the target live DNA information storage unit, thereby realizing a hierarchical retrieval method that is compatible with the hierarchical storage structure of database array-storage disk-live DNA information storage unit, which is conducive to accurately locating the target storage unit corresponding to the digital information to be retrieved.

[0046] In one embodiment of the present invention, the hierarchical live DNA data storage system further includes an engineered bacteria release module and an information reading module. The engineered bacteria release module is used to release engineered bacteria from the target live DNA information storage unit; the information reading module is used to extract information plasmids from the released engineered bacteria, perform sequencing and decoding on the extracted information plasmids, and obtain the digital information to be retrieved.

[0047] In this embodiment, the engineered bacteria in the target live DNA information storage unit are encapsulated in a microsphere encapsulation matrix. Therefore, before information reading, the engineered bacteria need to be released from the target live DNA information storage unit via the engineered bacteria release module. Subsequently, the information reading module extracts the information plasmid from the released engineered bacteria and performs sequencing and decoding on the DNA coding sequence carried in the information plasmid to recover the corresponding digital information to be retrieved. With the above settings, after locating the target live DNA information storage unit, the system can further realize the actual reading of the information stored therein.

[0048] In one embodiment of the present invention, the hierarchical live DNA data storage system further includes a biological processing module and a repackaging module. The biological processing module is used to culture and amplify the released engineered bacteria to obtain amplified engineered bacteria; the repackaging module is used to mix the amplified engineered bacteria with a packaging matrix and repackage it into a new live DNA information storage unit, and then return the new live DNA information storage unit to the target storage disk.

[0049] In this embodiment, the biological processing module is used to culture and amplify the released engineered bacteria after information reading, thereby increasing the number of engineered bacteria carrying the target information. The repackaging module is used to repackage the amplified engineered bacteria to form new live DNA information storage units and replenish them to the target storage disk. In this way, the system can not only complete information reading, but also re-form the corresponding storage units after reading and replenish them to the original storage location. Through the above settings, it is beneficial to maintain the continuous existence of the corresponding live DNA information storage units in the target storage disk.

[0050] In one embodiment of the present invention, the biological processing module is further configured to rewrite the DNA coding sequence on the information plasmid in the released engineered bacteria in situ, and to culture and amplify the engineered bacteria after the in situ rewriting to obtain the rewritten and amplified engineered bacteria; the repackaging module is further configured to mix the rewritten and amplified engineered bacteria with the packaging matrix and repackage it into a new live DNA information storage unit, and to place the new live DNA information storage unit into a predetermined storage disk.

[0051] In this embodiment, the biological processing module, in addition to culturing and amplifying the released engineered bacteria, can also perform in-situ rewriting of the DNA coding sequence on the information plasmid, thereby updating the stored information. After in-situ rewriting, the rewritten engineered bacteria are then cultured and amplified to obtain engineered bacteria carrying the updated information. The repackaging module further repackages the rewritten and amplified engineered bacteria into a new live DNA information storage unit and places it into a predetermined storage disk. The predetermined storage disk can be the original target storage disk or another storage disk redefined based on the rewritten information content. Through the above settings, the system can not only complete information reading but also repackage and reorganize the storage of the corresponding storage unit after information updates.

[0052] In one embodiment of the present invention, the engineered bacteria also carries an editing helper plasmid, which is used to target and cut old information sites on the information plasmid and to write new information fragments in situ through homologous recombination, thereby realizing the in situ rewriting of the information stored on the information plasmid; the biological processing module is specifically used to perform in situ rewriting of the DNA coding sequence on the information plasmid in the released engineered bacteria based on the editing helper plasmid in the released engineered bacteria.

[0053] In an optional embodiment of the present invention, there may be one or more editing helper plasmids.

[0054] In this embodiment, the edit-aid plasmid provides editing support for rewriting the DNA coding sequence on the information plasmid. Specifically, the edit-aid plasmid can identify and target old information sites on the information plasmid, and after cutting, write new information fragments into the corresponding positions through homologous recombination, thereby updating the original DNA coding sequence. The biological processing module can perform in-situ rewriting of the DNA coding sequence on the information plasmid based on the edit-aid plasmid, enabling the system to have a relatively clear information update capability. Through the above settings, the hierarchical live DNA data storage system can further achieve targeted modification of the stored content based on reading the original information.

[0055] In one embodiment of the present invention, the engineered bacteria release module specifically releases the engineered bacteria in the target living DNA information storage unit through solvent dissolution or physical triggering.

[0056] In this embodiment, since the engineered bacteria are encapsulated in a microsphere encapsulation matrix, they need to be released from their encapsulation state before information reading or subsequent biological processing. The engineered bacteria release module can release the encapsulated engineered bacteria by dissolving them in a solvent to break down the microsphere encapsulation matrix; alternatively, it can release the engineered bacteria from the target live DNA information storage unit from the microsphere encapsulation matrix through a physical triggering method. This physical triggering method can be, for example, pressure triggering, shearing triggering, vibration triggering, or other physical methods capable of decapsulation. With the above setup, operable engineered bacteria samples can be provided for subsequent processes such as plasmid extraction, sequencing decoding, culture amplification, and in-situ rewriting.

[0057] In one embodiment of the present invention, the hierarchical live DNA data storage system further includes a freeze-drying module and a rehydration module. The freeze-drying module is used to freeze-dry the live DNA information storage units in the database array, so that the live DNA information storage units are preserved in a freeze-dried state; the rehydration module is used to rehydrate the freeze-dried live DNA information storage units.

[0058] In this embodiment, the freeze-drying module is used to convert the live DNA information storage units in the database array from a conventional hydrated state to a freeze-dried state, thereby improving their preservation stability and facilitating long-term storage. The rehydration module is used to rehydrate the freeze-dried live DNA information storage units when retrieval, reading, or subsequent processing is required, restoring them to a detectable and processable state.

[0059] In this embodiment, the rehydrated live DNA information storage unit can still be identified and selectively extracted based on its detectable phenotypic signals. Thus, freeze-drying does not affect subsequent identification and retrieval of the target live DNA information storage unit using detectable phenotypic signals. Through the above configuration, the hierarchical live DNA data storage system can meet both long-term preservation needs and subsequent retrieval requirements.

[0060] In one embodiment of the present invention, the detectable phenotypic signal is a phenotypic signal that can be identified by flow cytometry detection and / or flow cytometry sorting; the phenotypic indexing unit is used to regulate fluorescent protein expression to form the detectable phenotypic signal.

[0061] In this embodiment, the phenotypic indexing unit can regulate the expression state of fluorescent proteins in engineered bacteria, enabling different live DNA information storage units to exhibit detectable fluorescent phenotypic signals. Thus, these detectable phenotypic signals can not only serve as a retrieval index for DNA coding sequences in information plasmids, but can also be identified through flow cytometry detection and / or flow cytometry sorting, facilitating the screening and location of target live DNA information storage units. Through the above configuration, the retrieval index in the hierarchical live DNA data storage system can be both detectable and sortable, thereby improving the identification efficiency of target live DNA information storage units.

[0062] In one embodiment of the present invention, the engineered bacteria also carries an editing helper plasmid, which can target and cut old information sites on the information plasmid and realize the in-situ writing of new information fragments through homologous recombination, thereby realizing the in-situ rewriting of the information stored on the information plasmid; the information plasmid and the editing helper plasmid constitute a mutually orthogonal dual plasmid system.

[0063] In this embodiment, the information plasmid is mainly used to carry the DNA coding sequence corresponding to the digital information, and the editing helper plasmid is mainly used to provide the auxiliary functions required for editing the DNA coding sequence. By setting the two as a mutually orthogonal dual plasmid system, the information storage function and the editing helper function can be separated, which is beneficial to achieve targeted modification of the target sequence on it while maintaining the information plasmid as the main storage carrier.

[0064] The edit-aid plasmid can target and cut old information sites on the information plasmid, and in conjunction with homologous recombination, achieve in-situ writing of new information fragments, thereby updating the original stored information. This setup provides a relatively clear basis for information rewriting in the hierarchical live DNA data storage system.

[0065] In one embodiment of the present invention, the living DNA information storage unit is a bacterial microsphere, which is formed from a crosslinkable matrix material. The bacterial microsphere is formed from droplets carrying the engineered bacteria formed by a droplet microfluidic two-phase system, which are then solidified or crosslinked.

[0066] In this embodiment, the live DNA information storage unit is in the form of bacterial-loaded microspheres. The crosslinkable matrix material is used to form an encapsulation and support structure for the engineered bacteria, enabling the engineered bacteria to be encapsulated and stably exist in the form of microspheres. By using a crosslinkable matrix material, bacterial-loaded microspheres with a certain structural stability can be obtained after droplet formation through solidification or crosslinking.

[0067] In this embodiment, the bacterial-loaded microspheres can be prepared using a droplet microfluidic two-phase system. Specifically, droplets carrying the engineered bacteria can first be formed in the droplet microfluidic two-phase system, and then the droplets can be solidified or cross-linked to form bacterial-loaded microspheres. The live DNA information storage unit prepared in the above manner has good modularity and microsphere characteristics, making it easy to set up and organize on storage disks. Through the above setup, a live DNA information storage unit format suitable for array-based arrangement and subsequent retrieval processing can be provided for the hierarchical live DNA data storage system.

[0068] In one embodiment of the present invention, the live DNA information storage unit includes a microsphere encapsulation matrix and engineered bacteria encapsulated within the microsphere encapsulation matrix. The engineered bacteria carry an information plasmid, which includes a phenotypic index unit and an information sequence unit. The information sequence unit stores the DNA coding sequence corresponding to the digital information to be stored; the phenotypic index unit generates a detectable phenotypic signal, which serves as a retrieval index for the DNA coding sequence.

[0069] In one embodiment of the present invention, the detectable phenotypic signal is a phenotypic signal that can be identified by flow cytometry detection and / or flow cytometry sorting. Specifically, the phenotypic index unit is used to regulate the expression of fluorescent proteins to form a detectable phenotypic signal; the detectable phenotypic signal is a monochromatic fluorescence signal, a two-color fluorescence combination signal, or a three-color fluorescence combination signal. This design allows different information files to be labeled with different fluorescence combinations, facilitating rapid retrieval and sorting in large-scale storage units. The phenotypic signal is preferably fluorescent protein expression, such as EGFP, mCherry, TagBFP, etc. The phenotypic index unit, as the Key part, logically forms a decoupled Key-Value structure with the Value part, facilitating subsequent non-destructive retrieval.

[0070] In one embodiment of the present invention, the engineered bacteria also carry an editing helper plasmid. The editing helper plasmid is used to target and cut old information sites on the information plasmid and to write new information fragments in situ through homologous recombination, thereby achieving in-situ rewriting of the information stored on the information plasmid. The information plasmid and the editing helper plasmid constitute a mutually orthogonal dual plasmid system. This dual plasmid system design makes the information storage and editing functions independent of each other, avoiding interference between functions.

[0071] In one embodiment of the present invention, the editing helper plasmid includes a nuclease expression module, a homologous recombination module, and an inducible expression control module. The nuclease expression module expresses a nuclease; the homologous recombination module mediates homologous recombination; and the inducible expression control module keeps the nuclease expression module and homologous recombination module in a closed or low-leakage expression state in a non-rewriting state, and initiates their expression under inducing conditions to target and cleave old information sites on the information plasmid and achieve in-situ writing of new information fragments through homologous recombination. This design ensures that the editing system is in a closed state when editing is not required, reducing the metabolic burden on the host cell and improving system stability.

[0072] In one embodiment of the present invention, the nuclease expression module is used to express the Cas12a nuclease, and the homologous recombination module is a λ-Red homologous recombination module. The Cas12a nuclease has efficient and precise DNA cutting capabilities, while the λ-Red homologous recombination system can effectively promote homologous recombination of DNA fragments. The combination of the two can achieve precise rewriting of information stored on information plasmids.

[0073] In one embodiment of the present invention, the edit helper plasmid preferably carries a Cas12a nuclease and a λ-Red homologous recombination system, and is equipped with an inducible expression control module to keep the editing system off or with low leakage in the non-rewriting state, thereby reducing long-term metabolic burden. When rewriting of the target file is required, after completing physical addressing and obtaining the target unit, the edit helper plasmid is induced to express: Cas12a, guided by crRNA / gRNA, targets and cuts the old information sites on the information plasmid, and simultaneously triggers λ-Red-mediated homologous recombination in the presence of donor DNA, thereby achieving site-specific erasure of old information and in-situ writing of new information.

[0074] In one embodiment of the present invention, the live DNA information storage unit is a bacterial microsphere, which is formed by solidifying or cross-linking droplets carrying engineered bacteria in a droplet microfluidic two-phase system. This microsphere encapsulation method physically isolates engineered bacteria carrying different information, avoiding population competition and database drift problems that may occur in mixed culture, while also facilitating subsequent storage, transportation, and retrieval operations.

[0075] Specifically, engineered bacteria carrying information plasmids are mixed with crosslinkable matrix materials, and droplets are formed using droplet microfluidics, followed by solidification or crosslinking treatment. The solidification method can employ one or more of photocrosslinking, thermogelation, ionic crosslinking, or chemical crosslinking. By adjusting the flow ratio of the dispersed phase to the continuous phase, the channel geometry, and the solidification conditions, the resulting microspheres can be controlled within the range of 20-100 μm, more preferably less than 60 μm, and exhibit a narrow particle size distribution.

[0076] In one embodiment of the present invention, the bacterial-carrying microspheres are formed from a crosslinkable matrix material, which includes at least one selected from gelatin, gelatin methacrylamide, alginate, hyaluronic acid derivatives, and polyethylene glycol hydrogel materials. These materials have good biocompatibility, providing a suitable microenvironment for the encapsulated engineered bacteria while maintaining the stability of the microsphere structure.

[0077] In one embodiment of the present invention, the microsphere encapsulation matrix contains engineered bacteria derived from the same engineered bacterial clone and carrying the same information plasmid. This design ensures the consistency of the information content stored in each microsphere unit and avoids reading errors caused by information mixing.

[0078] In one embodiment of the present invention, the phenotypic index unit and the information sequence unit are disposed on the same information plasmid. This design simplifies the system structure and ensures that the correspondence between the phenotypic index and the stored information is not lost due to plasmid separation.

[0079] In one embodiment of the present invention, the engineered bacterium is *Escherichia coli*. *Escherichia coli*, as a commonly used model organism, has advantages such as rapid growth, ease of genetic manipulation, and a wealth of molecular biology tools, making it an ideal host for constructing a living DNA information storage system.

[0080] In one embodiment of the present invention, the present invention specifically uses the Escherichia coli TOP10 strain. The Escherichia coli TOP10 strain used in this scheme was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0081] Based on the same inventive concept, another aspect of the present invention provides an operating method for a hierarchical live DNA data storage system, applicable to the hierarchical live DNA data storage system. Since the principle underlying the problem solved by the operating method of the hierarchical live DNA data storage system is similar to that of the hierarchical live DNA data storage system, embodiments of the operating method of the hierarchical live DNA data storage system can be found in the embodiments of the hierarchical live DNA data storage system, and repeated details will not be elaborated further.

[0082] like Figure 5 As shown, in one embodiment of the present invention, the operation method of the hierarchical live DNA data storage system of the present invention includes steps S101 to S103.

[0083] Step S101: When receiving an information retrieval instruction, the retrieval module first determines the target storage disk corresponding to the digital information to be retrieved based on the preset data index information. Then, by matching the detectable phenotypic signals of each live DNA information storage unit in the target storage disk with the target detectable phenotypic signals corresponding to the digital information to be retrieved, the target live DNA information storage unit corresponding to the digital information to be retrieved is determined. Step S102: Release the engineered bacteria in the target live DNA information storage unit through the engineered bacteria release module; Step S103: Extract information plasmids from the released engineered bacteria using the information reading module, and sequence and decode the extracted information plasmids to obtain the digital information to be retrieved.

[0084] In one embodiment of the present invention, the operation method of the hierarchical live DNA data storage system of the present invention further includes: The released engineered bacteria are cultured and amplified using a biological processing module to obtain amplified engineered bacteria. The amplified engineered bacteria are mixed with the encapsulation matrix and repackaged into a new live DNA information storage unit through the repackaging module, and the new live DNA information storage unit is then returned to the target storage disk.

[0085] In one embodiment of the present invention, the operation method of the hierarchical live DNA data storage system of the present invention further includes: The DNA coding sequence on the information plasmid in the released engineered bacteria is rewritten in situ using a biological processing module, and the engineered bacteria that have completed the in situ rewriting are cultured and amplified to obtain the rewritten and amplified engineered bacteria. The repackaged module mixes the rewritten and amplified engineered bacteria with the packaging matrix and repackages them into a new live DNA information storage unit, which is then placed into a predetermined storage disk.

[0086] In one embodiment of the present invention, a hierarchical live DNA data storage system and its hierarchical retrieval and closed-loop operation mode are provided. The hierarchical live DNA data storage system, by constructing a multi-level storage architecture and corresponding retrieval, reading, processing, and replenishment processes, achieves the orderly organization, target location, and information reading and updating of live DNA information storage units.

[0087] In this embodiment, the hierarchical live DNA data storage system comprises a three-tiered structure. The bottom layer consists of live DNA information storage units. Each live DNA information storage unit can be an engineered live storage microsphere (ELMM), comprising a microsphere encapsulation matrix and engineered bacteria encapsulated within the microsphere encapsulation matrix. The engineered bacteria can be, for example, *Escherichia coli*, serving as the information storage substrate. The engineered bacteria carry an information plasmid, which includes a phenotypic index unit and an information sequence unit. The information sequence unit stores the DNA coding sequence corresponding to the digital information to be stored, and the phenotypic index unit generates a detectable phenotypic signal, thereby establishing a correspondence between the DNA coding sequence and the observable phenotype. Figure 1 This schematically illustrates the correspondence between information sequences and detectable phenotypic signals in a living DNA information storage unit.

[0088] In some implementations, a standardized information plasmid library can be pre-constructed, and digital information can be encoded and written into the information sequence units within the information plasmid. The phenotypic indexing unit may include a tunable fluorescent protein expression module, which, under the action of an inducible regulatory circuit, generates a programmable and detectable phenotypic signal, so that the living DNA information storage unit, while carrying the DNA coding sequence, possesses an identifiable and distinguishable indexing basis.

[0089] In some embodiments, the engineered bacteria may further carry an editing helper plasmid, thereby forming a dual plasmid system orthogonal to the information plasmid. The editing helper plasmid can be used to express a CRISPR-Cas12a and λ-Red recombination module to achieve site-specific cleavage and in-situ rewriting of the DNA coding sequence on the information plasmid. Specifically, Cas12a can be guided by crRNA to recognize the target sequence on the information plasmid and, in the presence of donor DNA, trigger λ-Red-mediated homologous recombination to write new information fragments into the target site, thus completing the information update operation.

[0090] In some embodiments, the live DNA information storage unit can be prepared using droplet microfluidic technology. For example, a bacterial suspension carrying the engineered bacteria can be mixed with a matrix material and then microfluidically encapsulated, wherein the concentration of the engineered bacteria suspension can be, for example, at 10. 8 -10 10 The concentration of the matrix material is on the order of magnitude, for example, about 5-20 wt%. After forming droplets carrying the engineered bacteria through a droplet microfluidic two-phase system, and then performing solidification or cross-linking treatment, uniform bacterial-carrying microspheres with a diameter of, for example, less than 60 μm can be formed, thereby achieving unit-level physical isolation and encapsulation protection of the engineered bacteria.

[0091] In this embodiment of the invention, the intermediate layer is a storage disk. The storage disk is used to organize and carry multiple live DNA information storage units, enabling batch setup and centralized management of these units. The top layer is a database array. The database array consists of multiple storage disks, forming a higher-level live DNA data storage structure. Through this setup, a hierarchical organizational structure of live DNA information storage units—storage disks—database array can be formed, thereby expanding live DNA information from unit-level carrying to disk-level organization and array-level management. In some implementations, the layers can be connected via identification codes and physical channels to achieve hierarchical data management.

[0092] In this embodiment of the invention, the hierarchical live DNA data storage system can employ a hierarchical retrieval method adapted to the aforementioned hierarchical structure. Specifically, macroscopic positioning is performed first. The system can determine the target storage disk or target region corresponding to the digital information to be retrieved based on the user's query command, combined with high-level directory information, identification information, or preset data index information. For example, storage disks containing the target data category can be quickly located using microfluidic coding or visual recognition tags. Subsequently, microscopic screening is performed within the selected target storage disk. Specifically, the live DNA information storage units in the target storage disk can be mixed and suspended, and identification and screening can be performed based on the detectable phenotypic signals of the live DNA information storage units. In some embodiments, fluorescence-assisted sorting (FAS) or similar methods can be used to screen the live DNA information storage units one by one based on fluorescent labels, thereby identifying the target live DNA information storage units containing the target DNA coding sequence. Through the above hierarchical retrieval method, it is not necessary to perform unified sequencing processing on all storage units in the entire database array; instead, the target storage disk can be located first, and then the target live DNA information storage unit can be located within that target storage disk, thereby improving the targeting of information access.

[0093] In this embodiment of the invention, after the target live DNA information storage unit is located, the engineered bacteria in the target live DNA information storage unit can be further released through the engineered bacteria release module. Specifically, the microsphere encapsulation matrix can be destroyed by solvent dissolution or physical triggering to release the encapsulated engineered bacteria into a subsequent processing area, such as a biochemical processing channel. Subsequently, the information plasmid can be extracted from the released engineered bacteria through the information reading module, and the extracted information plasmid can be sequenced and decoded to obtain the digital information to be retrieved.

[0094] In this embodiment of the invention, after information reading is completed, the released engineered bacteria can be further processed by a biological processing module. For example, the target engineered bacteria can be cultured and amplified to obtain amplified engineered bacteria, thereby achieving information copying; alternatively, gene editing techniques can be used to modify the DNA coding sequence on the information plasmid to achieve information rewriting. The rewritten engineered bacteria can then be further cultured and amplified to obtain rewritten and amplified engineered bacteria.

[0095] In this embodiment of the invention, the amplified or rewritten amplified engineered bacteria can be further mixed with the microsphere encapsulation matrix through a re-encapsulation module and re-encapsulated to form a new live DNA information storage unit. After forming the new live DNA information storage unit, it can be placed back into the corresponding storage disk position in the original system to achieve replenishment; or, in some embodiments, the new live DNA information storage unit can also be placed into a predetermined storage disk according to the rewritten information content, index relationship, or storage plan to achieve reorganized storage.

[0096] In this embodiment of the invention, the aforementioned processes of retrieval, release, information reading, biological processing, repackaging, and replenishment can be completed collaboratively by microfluidic devices and automated instruments, thereby forming a closed-loop operation process from data reading to information updating and then to repackaging and storage. Through the above configuration, the hierarchical live DNA data storage system can not only achieve hierarchical positioning and information reading of target live DNA information storage units, but also continue to complete engineered bacterial amplification, information rewriting, and repackaging and replenishment of new storage units after reading.

[0097] This invention proposes a hierarchical live DNA data storage system and its related implementation methods, focusing on hierarchical storage, graded retrieval, information updating, and automated operation of live DNA information. The main technical points of this invention are further summarized below.

[0098] 1. Hierarchical Live DNA Data Storage Architecture. This invention constructs a hierarchical storage architecture consisting of a live DNA information storage unit layer, a storage disk layer, and a database array layer. The live DNA information storage unit serves as the basic storage unit, mounted on the storage disk. Multiple storage disks further constitute a database array, thus forming a DNA data storage system with a hierarchical organizational relationship. Through this configuration, live DNA information can be expanded from unit-level storage to disk-level organization and array-level management, thereby improving the systematic storage capability of live DNA information.

[0099] 2. Engineered Living DNA Information Storage Unit Structure. In this invention, the living DNA information storage unit is an engineered living storage microsphere (ELMM). The living DNA information storage unit includes an encapsulation matrix, engineered microbial cells encapsulated within the encapsulation matrix, and information plasmids stored within the engineered microbial cells. The information plasmids are used to store DNA coding sequences corresponding to digital information and can achieve observable signal output through phenotypic expression modules within the cells, thus enabling the living DNA information storage unit to possess identifiable characteristics while carrying information.

[0100] 3. Key-Value Indexing Mechanism Based on Biological Phenotype. This invention constructs a phenotypic expression module in engineered microbial cells. This phenotypic expression module generates observable signals, which establish a correspondence with DNA coding sequences stored in information plasmids, thereby forming a key-value index structure for DNA information addressing and identification. Through this setup, the living DNA information storage unit not only carries the DNA coding sequence corresponding to digital information but also provides an indexing basis through detectable phenotypic signals, facilitating subsequent target identification and retrieval.

[0101] 4. Cross-scale hierarchical random access method. This invention also provides a hierarchical retrieval method suitable for the hierarchical live DNA data storage system. This retrieval method may include: first, macroscopic positioning using the identification information of the database array or storage disk to determine the storage disk where the target data is located; then, screening for corresponding live DNA information storage units within the target storage disk using phenotypic signal recognition technology; finally, DNA extraction, amplification, and / or sequencing of the screened target live DNA information storage units to read the target information. Through the above hierarchical retrieval method, layer-by-layer positioning and random access from the disk level to the unit level can be achieved.

[0102] 5. A cyclical update mechanism for the living DNA information storage unit. In this invention, for the target living DNA information storage unit obtained through retrieval, the cells within can be further cultured and amplified, gene-edited, and / or DNA rewritten to achieve the replication, updating, or modification of DNA information. The processed cells can also be repackaged to form new living DNA information storage units and returned to the hierarchical living DNA data storage system. Through the above settings, the living DNA information storage unit can continue to be used for information maintenance or information updating after information reading is completed.

[0103] 6. Automated Closed-Loop Operation Mode of DNA Storage System. In this invention, an automated operation process suitable for DNA data storage operations can be constructed. This automated operation process may include storage unit retrieval, cell release, biological processing, information amplification and / or editing, storage unit repackaging, and system replenishment, thereby forming a closed-loop operation mechanism for DNA data storage. Through the above settings, the system can continue to complete cell processing, information updating, and the formation and replenishment of new storage units after completing the retrieval and reading of target information.

[0104] 7. Microfluidic Preparation Method of Living DNA Information Storage Unit. In this invention, droplet microfluidic technology can be used to microscale encapsulate engineered microbial cells with an encapsulation matrix to prepare engineered living storage microspheres with uniform size. Specifically, droplets carrying the engineered microbial cells can be formed through a droplet microfluidic two-phase system, and then solidified or cross-linked to form living DNA information storage units. Through the above method, physical isolation and large-scale construction of DNA information units can be achieved.

[0105] 8. Automated DNA Storage Drive Device. This invention also includes an automated device for driving the operation of a DNA data storage system. The automated device may include a microfluidic processing module, a storage unit retrieval module, a biological processing module, and a storage unit repackaging module, for automatically retrieving, releasing, processing, repackaging, and replenishing living DNA information storage units. Through the above configuration, an automated drive foundation adapted to the operational flow of the hierarchical living DNA data storage system can be provided.

[0106] 9. Automated Platform System for Live DNA Data Storage. This invention can also construct an automated platform system for live DNA data storage. The automated platform system includes the aforementioned hierarchical live DNA data storage system and automated devices for driving the operation of the storage system. Through system-level control, the automated platform system can realize the writing, storage, retrieval, replication, updating, and recycling of DNA information, thereby improving the overall operational capability and scalability of the live DNA data storage system.

[0107] Figure 1 This section describes the construction, usage, and functions of an Elastic DNA Information Storage Unit (ELMM). Part A illustrates the design of the information storage strain and the preparation of the storage unit. Part B demonstrates the standardized reuse process of the storage system.

[0108] Figure 2 A schematic diagram of the hierarchical retrieval structure is shown, illustrating the hierarchical relationship between ELMM units, storage disks, and database arrays.

[0109] Figure 3This is a flowchart illustrating the automated operation of a DNA storage system, showing the processes of retrieval, release, amplification / editing, repackaging, and replenishment.

[0110] The present solution will be further explained below with reference to specific embodiments.

[0111] Example 1: This embodiment demonstrates a method for preparing a live DNA information storage unit (ELMM). In this embodiment, Escherichia coli DH5α is used as the engineered bacterium, a pUC-based plasmid is used, and sodium alginate microspheres are used to prepare the live DNA information storage unit (ELMM) with a diameter of 30–50 μm.

[0112] Example 2: This embodiment conducted a retrieval experiment, using three-color fluorescent labeling and FACS sorting. Through actual experiments, the sorting accuracy of the proposed solution can reach 95%.

[0113] As can be seen from the above embodiments, compared with the prior art, the solution of the present invention has at least the following beneficial effects: 1. This invention constructs a hierarchical storage architecture consisting of a live DNA information storage unit layer, a storage disk layer, and a database array layer, which expands the live DNA information from unit-level carrying to disk-level organization and array-level management, thereby improving the systematic storage and large-scale organization capabilities of live DNA information.

[0114] 2. This invention introduces a phenotypic index unit into the living DNA information storage unit, which establishes a correspondence between the DNA coding sequence stored in the information plasmid and the detectable phenotypic signal. This enables the living DNA information storage unit to carry information while having an identifiable and distinguishable index basis, which is conducive to the rapid location and hierarchical retrieval of the target storage unit.

[0115] 3. By setting up modules for engineered bacteria release, information reading, biological processing, and repackaging, this invention can culture and amplify the released engineered bacteria after the target information is read, and repackage them to form new living DNA information storage units, thereby helping to maintain the continuous existence of the corresponding storage units; furthermore, by rewriting the DNA coding sequence on the information plasmid in situ, the storage information can also be updated and reorganized.

[0116] 4. By combining the hierarchical storage structure with the processes of retrieval, release, biological processing, repackaging, and replenishment, this invention can form a relatively complete closed-loop operation mechanism, which helps to reduce the complexity caused by manual decentralized operation and improve the continuity and overall coordination of the operation process of the live DNA data storage system.

[0117] 5. This invention uses a microsphere encapsulation matrix to encapsulate engineered bacteria, so that the live DNA information exists in a modular, microsphere form. It can also be combined with freeze-drying and rehydration methods to achieve long-term preservation and subsequent retrieval of live DNA information storage units, thereby improving the storage stability, manageability and subsequent flexibility of the system.

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hierarchical live DNA data storage system, characterized in that, This includes a database array, which consists of multiple storage disks, each of which is provided with multiple live DNA information storage units. The live DNA information storage unit includes a microsphere encapsulation matrix, which encapsulates engineered bacteria; the engineered bacteria carry an information plasmid, which includes a phenotypic index unit and an information sequence unit; the information sequence unit stores the DNA coding sequence corresponding to the digital information to be stored; the phenotypic index unit can generate a detectable phenotypic signal during detection, and the detectable phenotypic signal is used as a retrieval index for the DNA coding sequence.

2. The hierarchical live DNA data storage system according to claim 1, characterized in that, Also includes: The retrieval module is used to determine the target storage disk corresponding to the digital information to be retrieved based on preset data index information, and to determine the target live DNA information storage unit corresponding to the digital information to be retrieved by matching the detectable phenotypic signals of each live DNA information storage unit in the target storage disk with the target detectable phenotypic signals corresponding to the digital information to be retrieved.

3. The hierarchical live DNA data storage system according to claim 2, characterized in that, Also includes: An engineered bacteria release module is used to release engineered bacteria from the target live DNA information storage unit; The information reading module is used to extract information plasmids from the released engineered bacteria, sequence and decode the extracted information plasmids to obtain the digital information to be retrieved.

4. The hierarchical live DNA data storage system according to claim 3, characterized in that, Also includes: The biological processing module is used to culture and amplify the released engineered bacteria to obtain amplified engineered bacteria; The repackaging module is used to mix the amplified engineered bacteria with the packaging matrix and repackage them into a new live DNA information storage unit, and then return the new live DNA information storage unit to the target storage disk.

5. The hierarchical live DNA data storage system according to claim 4, characterized in that, The biological processing module is also used to rewrite the DNA coding sequence on the information plasmid in the released engineered bacteria in situ, and to culture and amplify the engineered bacteria after the in situ rewriting to obtain the rewritten and amplified engineered bacteria. The repackaging module is also used to mix the rewritten and amplified engineered bacteria with the packaging matrix and repackage them into a new live DNA information storage unit, and place the new live DNA information storage unit into a predetermined storage disk.

6. The hierarchical live DNA data storage system according to claim 5, characterized in that, The engineered bacteria also carry an editing helper plasmid, which can target and cut old information sites on the information plasmid and write new information fragments in situ through homologous recombination, thereby realizing the in situ rewriting of the information stored on the information plasmid. The biological processing module is specifically used to rewrite the DNA coding sequence on the information plasmid in the released engineered bacteria in situ, based on the editing helper plasmid in the released engineered bacteria.

7. The hierarchical live DNA data storage system according to claim 3, characterized in that, The engineered bacteria release module specifically releases the engineered bacteria in the target living DNA information storage unit through solvent dissolution or physical triggering.

8. The hierarchical live DNA data storage system according to claim 1, characterized in that, Also includes: The freeze-drying module is used to freeze-dry the live DNA information storage unit in the database array so that the live DNA information storage unit is preserved in a freeze-dried state. The rehydration module is used to rehydrate the freeze-dried live DNA information storage unit, wherein the freeze-dried live DNA information storage unit can still be identified and selectively extracted based on the detectable phenotypic signal after rehydration.

9. The hierarchical live DNA data storage system according to claim 1 or 2, characterized in that, The detectable phenotypic signal is a phenotypic signal that can be identified by flow cytometry detection and / or flow cytometry sorting; the phenotypic indexing unit is used to regulate fluorescent protein expression to form the detectable phenotypic signal.

10. The hierarchical live DNA data storage system according to claim 1, characterized in that, The engineered bacteria also carry an editing helper plasmid, which can target and cut old information sites on the information plasmid and write new information fragments in situ through homologous recombination, thereby realizing the in situ rewriting of the information stored on the information plasmid; the information plasmid and the editing helper plasmid constitute a mutually orthogonal dual plasmid system.

11. The hierarchical live DNA data storage system according to claim 1, characterized in that, The living DNA information storage unit is a bacterial microsphere, which is formed from a cross-linkable matrix material. The bacterial microsphere is formed from droplets carrying the engineered bacteria in a droplet microfluidic two-phase system, which are then solidified or cross-linked.

12. A method for operating a hierarchical live DNA data storage system, characterized in that, The method of operating the hierarchical live DNA data storage system as described in any one of claims 1 to 11 includes: Upon receiving an information retrieval instruction, the target storage disk corresponding to the digital information to be retrieved is first determined based on the preset data index information. Then, the target live DNA information storage unit corresponding to the digital information to be retrieved is determined by matching the detectable phenotypic signal of each live DNA information storage unit in the target storage disk with the target detectable phenotypic signal corresponding to the digital information to be retrieved. Release the engineered bacteria from the target living DNA information storage unit; Information plasmids are extracted from the released engineered bacteria, and the extracted information plasmids are sequenced and decoded to obtain the digital information to be retrieved.

13. The method of operating the hierarchical live DNA data storage system according to claim 12, characterized in that, Also includes: The released engineered bacteria were cultured and amplified to obtain the amplified engineered bacteria; The amplified engineered bacteria are mixed with the encapsulation matrix and repackaged into a new live DNA information storage unit, which is then returned to the target storage disk.

14. The method of operating the hierarchical live DNA data storage system according to claim 12, characterized in that, Also includes: The DNA coding sequence on the information plasmid in the released engineered bacteria was rewritten in situ, and the engineered bacteria that had completed the in situ rewriting were cultured and amplified to obtain the rewritten and amplified engineered bacteria. The rewritten and amplified engineered bacteria are mixed with the encapsulation matrix and repackaged into a new live DNA information storage unit, which is then placed into a predetermined storage disk.

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