Bifunctional engineered magnetotactic bacteria, construction methods, imaging methods for their implementation, and applications.

Abstract

This invention discloses a bifunctional engineered magnetotactic bacterium. The engineered magnetotactic bacterium is constructed using a recombinant PmamC-MamC-(Gly₄Ser)₃-MagLov fusion gene fragment to construct the expression plasmid pBBR1-MagLov-MamC. Using Magnetospirillum gryphiswaldense MSR-1 as a host, the recombinant expression plasmid pBBR1-MagLov-MamC is conjugated to obtain the bifunctional engineered magnetotactic bacterium MSR-1-MagLov capable of expressing the MagLov-MamC fusion protein. This invention also discloses the construction method and its applications. This invention overcomes the technical bias of exogenous proteins inhibiting magnetosome synthesis, achieving the integration of magnetotactic motility and magnetosensitive fluorescence.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and applied technology, and in particular to a bifunctional engineered magnetotactic bacterium, its construction method, its imaging method, and its applications. Background Technology

[0002] Magnetotactic bacteria are a class of prokaryotic microorganisms capable of biosynthesizing magnetosomes intracellularly. These magnetosomes, composed of magnetite nanocrystals and a phospholipid membrane, enable directional movement of the bacteria along a magnetic field and have been explored for targeted delivery, environmental microbial tracing, and micro / nanorobotics. However, magnetotactic bacteria themselves lack fluorescence signals. Traditional labeling methods using exogenous fluorescent dyes or ordinary fluorescent proteins have the following significant drawbacks: the fluorescence signal cannot be modulated by a magnetic field, making it impossible to distinguish specific signals from tissue autofluorescence and non-specific adsorption background, resulting in extremely low imaging signal-to-noise ratios; exogenous fluorescent labels are easily quenched and leaked, making long-term stable tracing impossible; heterologous expression of exogenous proteins in magnetotactic bacteria can interfere with the magnetosome biomineralization process, exhibiting a technical bias where expression inhibits magnetotactic activity; and there is a lack of dedicated expression systems for magnetically responsive fluorescent elements adapted to magnetotactic bacteria, resulting in low transformation efficiency and poor genetic stability.

[0003] Magnetoric fluorescent proteins (MFLs) are artificially engineered fluorescent proteins that can reversibly alter their fluorescence intensity under the influence of an external magnetic field, enabling remote, non-invasive, and deep-penetrating modulation of fluorescence signals. However, they lack magnetic targeting and active motility, exhibiting only passive diffusion and distribution, thus limiting their precise accumulation in lesions and significantly restricting their application in living organisms and complex systems. Currently, there are no publicly reported functional integrations of MFLs with magnetotactic bacteria, no dual-function living microbial system possessing both magnetic field-directed magnetotactic motility and reversible magnetic fluorescence switching, and no integrated magnetic field differential imaging and diagnostic / therapeutic application scheme based on such a system.

[0004] Therefore, developing a genetically engineered magnetotactic bacterium that simultaneously expresses magnetofluorescein and retains magnetotactic activity, and applying it in in vivo tracking, not only has urgent research value, but also good economic benefits and industrial application potential. This is the driving force and foundation for the completion of this invention. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art as mentioned above, the inventors conducted in-depth research and, after a great deal of creative work, completed this invention.

[0006] Specifically, the technical problem to be solved by this invention is to provide a bifunctional engineered magnetotactic bacteria, its construction method, its imaging method, and its applications, so as to overcome the technical bias of exogenous protein expression inhibiting the synthesis of magnetosomes in magnetotactic bacteria, and to achieve stable compatibility and synergistic expression of magnetically controlled fluorescent proteins and magnetotactic bacteria; to construct bifunctional engineered magnetotactic bacteria that simultaneously possess active magnetic field-targeted movement and magnetic field-controllable fluorescence switching; to provide a set of efficient, stable, and reproducible processes for the construction, transformation, screening, and fermentation of engineered bacteria; to establish a new method of magnetic field differential fluorescence imaging to achieve non-invasive visualization of deep tissues with high signal-to-noise ratio; to develop in-situ magnetically controlled fluorescent magnetosome probes to simplify the preparation process and improve biocompatibility; to clarify the specific tumor-suppressing effect of engineered bacteria in tumor targeted therapy, as well as the precise detection limit of heavy metal ion detection, and to explore new applications of this bifunctional system in in vivo tracing, integrated tumor diagnosis and treatment, environmental sensing, and other fields.

[0007] To solve the above-mentioned technical problems, the first technical solution of the present invention is: A bifunctional engineered magnetotactic bacterium, wherein the engineered magnetotactic bacterium is constructed by using a recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment to construct the expression plasmid pBBR1-MagLov-MamC, and using Magnetospirillumgryphiswaldense MSR-1 as the host, conjugating the recombinant expression plasmid pBBR1-MagLov-MamC to obtain a bifunctional engineered magnetotactic bacterium MSR-1-MagLov capable of expressing the MagLov-MamC fusion protein.

[0008] As an improvement, in the recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment, MagLov, as a magnetron fluorescent protein amino acid sequence, can be replaced with either MagGFP or MagCherry.

[0009] As an improvement, in the recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment, MamC, as the amino acid sequence of the magnetosome membrane anchoring protein, can also be replaced with any one of Mms13 or MamF.

[0010] As an improvement, the engineered magnetotactic bacteria contain a recombinant expression vector with a backbone of pBBR1MCS-2 or pBBR1MCS-5, and the vector contains a conjugation transfer element, an resistance selection marker, and a multiple cloning site; the resistance selection marker is a chloramphenicol resistance gene, a kanamycin resistance gene, or an ampicillin resistance gene.

[0011] As an improvement, the bifunctional engineered magnetotactic bacteria MSR-1-MagLov exhibits reversible down-regulation of fluorescence intensity by 40%-80% under a magnetic field of 50-1000 mT, with no significant attenuation of fluorescence intensity after at least 50 consecutive magnetic field switching cycles, and stable inheritance of exogenous genes without loss after at least 20 consecutive generations.

[0012] The second technical solution of the present invention provides a method for constructing the bifunctional engineered magnetotactic bacteria, comprising the following steps: S1. A fusion gene consisting of the wild-type MamC gene, the (Gly4Ser)3 coding sequence, and the codon-optimized MagLov gene was synthesized and inserted into an expression vector to obtain a recombinant expression vector. S2. Using an optimized conjugation transfer method, the recombinant expression vector was transformed into magnetotactic bacteria MSR-1 to obtain primary screen transformants; S3. A two-step screening method combining magnetic enrichment and fluorescence flow cytometry was used to screen for positive bifunctional engineered magnetotactic bacteria. S4. Co-fermentation culture of positive engineered bacteria to obtain engineered magnetotactic bacteria with stable magnetotactic activity and magneto-controlled fluorescence function.

[0013] As an improvement, in step S1, the gene sequence is designed and the codons are optimized: the MagLov coding sequence is optimized using magnetotactic bacteria preferred codons, and the natural sequence of the MamC gene is preserved; the Kozak ribosome binding sequence, the N-terminal signal peptide coding sequence, and the C-terminal stop codon are added, and the coding sequence of the flexible linker peptide (Gly4Ser)3 is inserted between the MamC and MagLov coding regions. EcoRI and Hind III restriction endonuclease sites are introduced at both ends of the gene, and finally the PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment is obtained. Then, the vector was digested and ligated: the synthesized fusion gene fragment and the broad host plasmid pBBR1MCS-2 were digested with restriction endonucleases EcoR I and Hind III, respectively. The digestion products were recovered by agarose gel electrophoresis and ligated using T4 DNA ligase. Finally, transformation and positive clone screening: The ligation product was transformed into E. coli DH5α competent cells, cultured, identified by PCR, and the plasmid was extracted after amplification and culture of positive clones to obtain the recombinant expression plasmid pBBR1-MagLov-MamC.

[0014] As an improvement, in step S2, the donor bacteria for conjugation transfer is Escherichia coli S17-1, the mixing ratio of donor bacteria to magnetotactic recipient bacteria is 1:1 to 3:1, the conjugation time is 4-8 hours, and the culture conditions are a micro-aerobic environment with an oxygen content of 1%-3% and a temperature of 28-30℃.

[0015] As an improvement, in step S3, magnetic enrichment screening is first performed. The screened transformants are inoculated into the culture medium. After cultivation, the bacterial solution is placed next to a permanent magnet with a magnetic field strength of 300 mT to enrich magnetic sedimentation positive strains. Then, the magnetic sedimentation positive strains are subjected to fluorescence flow cytometry screening. Single clones of bacteria with high fluorescence signals are collected, inoculated into liquid culture medium for expansion culture, and the target bifunctional engineered magnetotactic bacteria MSR-1-MagLov is obtained. Finally, the function of the engineered bacteria is verified to obtain strains that meet the requirements.

[0016] As an improvement, in step S4, the parameters for co-fermentation culture are: dissolved oxygen 1%-5%, iron source concentration 20-80 μM, and induced OD... 600 The concentration should be 0.6-0.9, pH 6.8-7.2, and the culture temperature 28-30℃.

[0017] As an improvement, the construction method also includes the preparation step of in-situ magnetotropic fluorescent magnetosome probe: collecting engineered magnetotactic bacteria after fermentation, lysing them under hypotonic conditions, and then rapidly separating them by a magnetic field to obtain a magnetosome-magnetotropic fluorescent protein composite probe wrapped in a natural biomembrane.

[0018] The third technical solution of the present invention provides a magnetic field differential fluorescence imaging method using bifunctional engineered magnetotactic bacteria, comprising the following steps: (1) Under conditions without an external magnetic field, the first fluorescence image of the sample to be tested or the test subject is acquired, and the engineered bacteria are in a high fluorescence state; (2) Apply an external magnetic field of 50-1000 mT and collect the second fluorescence image. The engineered bacteria are in a low fluorescence state. (3) Perform pixel-level difference operation between the first fluorescence image and the second fluorescence image to remove background autofluorescence interference and obtain high signal-to-noise ratio target fluorescence imaging results; the imaging method is applicable to cell samples, tissue sections or live animals and can achieve non-invasive fluorescence imaging of deep tissues with a depth of not less than 5 mm.

[0019] The fourth technical solution of the present invention provides the application of bifunctional engineered magnetotactic bacteria, including its application in the preparation of in vivo tracer reagents, its application in the preparation of integrated tumor diagnosis and treatment agents, and its application in the preparation of whole-cell biosensors.

[0020] As an improvement, in the application of the preparation of in vivo tracer reagents, the in vivo tracer reagents are used for dynamic tracing of intestinal flora, microbial tracing of tumor microenvironment, or targeted tracing of inflammatory sites. The engineered bacteria are targeted and enriched through an external magnetic field, and non-invasive, visual, long-term tracking is achieved through magnetically controlled fluorescence signals.

[0021] As an improvement, in the application of integrated tumor diagnosis and treatment formulations, the engineered magnetotactic bacteria serve as drug carriers to load anti-tumor drugs, immune agonists, or gene therapy drugs, achieving targeted delivery to lesions under magnetic field drive. When combined with magnetic field-targeted drug delivery, the tumor inhibition rate in a 4T1 breast cancer nude mouse model can reach 66.67%. Furthermore, the bacterial colonization, drug release, and therapeutic effect can be monitored in real time through changes in magnetically controlled fluorescence signals, thus completing the integration of delivery, tracing, and efficacy evaluation.

[0022] As an improvement, in the application of whole-cell biosensors, the whole-cell biosensor is used to detect heavy metal cadmium ions, with a linear detection range of 0.1-10 μM, a detection limit as low as 0.08 μM, and a linear correlation coefficient R²=0.992. The fluorescence difference ΔF is controlled by a magnetic field to reflect the target concentration, and the rapid enrichment and separation of the sensing bacteria are achieved by using a magnetic field.

[0023] After adopting the above technical solution, the beneficial effects of the present invention are: This invention integrates magnetotactic motility and magneto-controlled fluorescence, overcoming the core deficiency of existing technologies with limited functionality. Magnetic field differential imaging significantly improves the signal-to-noise ratio, enabling high-resolution, non-invasive tracing of deep tissues with statistically significant results. The transformation, screening, and fermentation processes are comprehensively optimized, resulting in high efficiency, strong stability, and suitability for large-scale production. It supplements clearly defined data on tumor inhibition rates and heavy metal detection limits, covering multiple scenarios including in vivo tracing, targeted therapy, and biosensing, possessing extremely high research and clinical translational value. Overcoming the technical bias of exogenous proteins inhibiting magnetosome synthesis, it explicitly adopts the wild-type MamC gene and codon-optimized standard MagLov sequence, providing a new paradigm for the engineering of magnetotactic bacteria. The in-situ probe preparation process simplifies the process, reduces production costs, and improves product consistency and biocompatibility. Attached Figure Description

[0024] Figure 1 Schematic diagram of the structure of the magnetically controlled fluorescence fusion gene and the core function of the bifunctional engineered bacteria; Figure 2 Physical map of recombinant expression plasmid pBBR1-MagLov-MamC; Figure 3 Transmission electron microscopy (TEM) characterization of the bifunctional engineered magnetotactic bacteria MSR-1-MagLov; Figure 4 Fluorescence regulation curve of the magnetic field response of the magnetofluorescent protein MagLov; Figure 5 Schematic diagram of the principle and processing flow of magnetic field differential fluorescence imaging; Figure 6 Cell-level magnetic field differential fluorescence imaging effect; Figure 7Image of magnetic field differential fluorescence imaging of live tumor tissue; Figure 8 Characterization diagram of in-situ magnetically controlled fluorescent magnetosome probe; Figure 9 Standard curve of the response of engineered bacteria whole-cell sensor to Cd²⁺ concentration. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. However, the uses and purposes of these exemplary embodiments are only for illustrating the present invention and do not constitute any limitation on the actual scope of protection of the present invention, nor are they intended to limit the scope of protection of the present invention to these embodiments. Furthermore, experimental methods not specifically specified in the embodiments are performed according to conventional molecular biology, microbial culture and fermentation, and fluorescence imaging experimental conditions in the art, or according to the conditions recommended by reagent and instrument manufacturers.

[0026] A bifunctional engineered magnetotactic bacterium, wherein the engineered magnetotactic bacterium is constructed by using a recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment to construct the expression plasmid pBBR1-MagLov-MamC, and using Magnetospirillumryphiswaldense MSR-1 as the host, conjugating the recombinant expression plasmid pBBR1-MagLov-MamC to obtain a bifunctional engineered magnetotactic bacterium MSR-1-MagLov capable of expressing the MagLov-MamC fusion protein. Furthermore, the bifunctional engineered magnetotactic bacterium MSR-1-MagLov is required to exhibit a reversible downregulation of fluorescence intensity of 40%-80% under a magnetic field of 50-1000 mT, with no significant attenuation of fluorescence intensity after at least 50 continuous magnetic field switching modulations, and stable inheritance of the exogenous gene without loss after at least 20 consecutive generations.

[0027] In the recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment, MagLov, as the gene fragment encoding the amino acid sequence of a magnetron fluorescent protein, can be replaced with either MagGFP or MagCherry. MamC, as the gene fragment encoding the amino acid sequence of a magnetosome membrane anchoring protein, can also be replaced with either Mms13 or MamF. The engineered magnetotactic bacteria contain a recombinant expression vector (plasmid) with a backbone of pBBR1MCS-2 or pBBR1MCS-5. The vector contains a conjugation transfer element, an resistance selection marker, and a multiple cloning site; the resistance selection marker is a chloramphenicol resistance gene, a kanamycin resistance gene, or an ampicillin resistance gene.

[0028] In the subsequent construction methods of this embodiment, MagLov, MamC, pBBR1MCS-2, and chloramphenicol resistance gene were selected respectively.

[0029] In this invention, the magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1, Magnetospirillum magneticum AMB-1, Escherichia coli DH5α, and Escherichiacoli S17-1 are all publicly available type strains; the plasmids pBBR1MCS-2 and pBBR1MCS-5 are commercially available broad-host vectors; the MagLov magnetofluorescent protein gene is a standard sequence published in the literature, which was optimized for codon bias in magnetotactic bacteria and synthesized by a biotechnology company; the MamC gene is the wild-type sequence of Magnetospirillum gryphiswaldense MSR-1; and the reagents and culture media are all commercially available analytical grade or biochemically pure products.

[0030] Example 1: Synthesis of Magnetically Controlled Fluorescent Fusion Gene and Construction of Recombinant Expression Vector This embodiment uses the construction of the PmamC-MamC-(Gly4Ser)3-MagLov fusion gene and the recombinant plasmid pBBR1-MagLov-MamC as an example for illustration.

[0031] Gene sequence design and codon optimization: Based on the wild-type MamC gene sequence of Magnetospirillumgryphiswaldense MSR-1 in the NCBI database and the publicly available standard MagLov magnetoresistive fluorescent protein amino acid sequence, the MagLov coding sequence was optimized using magnetotactic bacteria preferred codons, preserving the natural MamC gene sequence. The Kozak ribosome binding sequence, the N-terminal signal peptide coding sequence, and the C-terminal stop codon were added. The coding sequence of the flexible linker peptide (Gly4Ser)3 was inserted between the MamC and MagLov coding regions. EcoRI and HindIII restriction endonuclease sites were introduced at both ends of the gene, respectively. Finally, the full-length fusion gene sequence was synthesized by a biotechnology company (the synthesis method and gene fragment acquisition methods are existing technologies and will not be described here).

[0032] Vector digestion and ligation: The synthesized fusion gene fragment and the broad-host plasmid pBBR1MCS-2 were double-digested with restriction endonucleases EcoRI and HindIII, respectively. The digestion system was as follows: plasmid / gene fragment 2 μg, 10×QuickCutBuffer 5 μL, EcoRI 1 μL, HindIII 1 μL, water to a final volume of 50 μL, and digested at 37℃ for 30 min. After recovery of the digested products by agarose gel electrophoresis, ligation was performed using T4 DNA ligase. The ligation system was as follows: linearized vector 1 μL, fusion gene fragment 3 μL, 10×T4 Buffer 1 μL, T4 DNA Ligase 1 μL, water to a final volume of 10 μL, and ligated overnight at 16℃.

[0033] Transformation and positive clone screening: The ligation product was transformed into *E. coli* DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, rapidly incubated on ice for 5 min, and then recovered in LB liquid medium for 1 h. The cells were then plated on LB agar plates containing 34 μg / mL chloramphenicol and incubated upside down at 37℃ for 12–16 h. Single colonies were picked for colony PCR identification. After amplification and culture of positive clones, plasmids were extracted, verified by EcoRI / HindIII double digestion, and sent to a sequencing company for full-length sequencing. Sequencing results showed that the fusion gene sequence was completely consistent with the design, with no frameshift mutations or base mismatches, and the recombinant expression plasmid pBBR1-MagLov-MamC was successfully obtained.

[0034] See Figure 1 The diagram illustrates the structure of the magnetofluorescent fusion gene and the core function of the bifunctional engineered bacteria. From top to bottom, the diagram shows the complete components of the fusion gene described in this invention, including the endogenous promoter PmamC of magnetotactic bacteria, the signal peptide coding sequence, the MamC magnetosome membrane protein coding region, the flexible linker peptide (Gly4Ser)3 coding region, the MagLov magnetofluorescent protein coding region, and the transcription terminator. Below, the diagram shows the intracellular magnetosome chain structure of the bifunctional engineered magnetotactic bacteria, the magnetic field-driven magnetotactic movement mode, and the effect of the magnetic field regulating the fluorescence switch, visually demonstrating the dual-functional characteristics of the fusion gene design and the engineered bacteria. Figure 2 The physical map of the recombinant expression plasmid pBBR1-MagLov-MamC shows the restriction enzyme sites, the chloramphenicol resistance gene Cmʳ (resistance selection marker), the origin of replication, and the position and orientation of the fusion gene expression elements of the broad-host recombinant plasmid constructed in this invention. The EcoRI and HindIII double restriction sites are marked, clearly reflecting the construction structure and key functional regions of the recombinant vector.

[0035] Example 2: Construction, transformation and screening of bifunctional engineered magnetotactic bacteria In this embodiment, MSR-1 was used as the host to construct a bifunctional engineered magnetotactic bacterium expressing the MagLov-MamC fusion protein.

[0036] Culture of donor and recipient bacteria: The recombinant plasmid pBBR1-MagLov-MamC constructed in Example 1 was transformed into Escherichia coli S17-1 competent cells to obtain donor bacteria S17-1 / pBBR1-MagLov-MamC, which were then inoculated into LB medium containing chloramphenicol and cultured at 37°C and 200 rpm until OD. 600 =0.6–0.8. Magnetotactic bacteria MSR-1 were inoculated into MSGM liquid medium and cultured under microaerobic conditions (O2 2%, 30°C, 150 rpm) until OD... 600 =0.6–0.9, as recipient bacteria.

[0037] Optimized conjugation transfer transformation: Donor and recipient bacterial cultures were mixed at a volume ratio of 2:1, centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the culture was resuspended twice with sterile physiological saline, finally resuspended in 100 μL of physiological saline. The bacterial culture was added dropwise to antibiotic-free MSGM plates lined with a 0.22 μm sterile filter membrane and incubated under microaerophilic conditions (O2 2%) at 30℃ for 6 h. The bacterial cells on the filter membrane were washed off with sterile physiological saline, serially diluted, and plated onto selective MSGM plates containing chloramphenicol 34 μg / mL and nalidixinone acid 25 μg / mL. The plates were incubated microaerophilically for 7–10 days to obtain the initial transformants.

[0038] Two-step screening using magnetic enrichment and fluorescence flow cytometry: For primary magnetic enrichment screening, transformants were inoculated into MSGM liquid medium containing resistance. After 48 h of culture, the bacterial suspension was placed near a permanent magnet with a magnetic field strength of 300 mT for 30 min for enrichment. The supernatant containing unenriched bacteria was aspirated, retaining magnetically sedimentation-positive strains and removing negative strains with magnetosome synthesis defects. For secondary fluorescence flow cytometry screening, the magnetically enriched bacteria were washed and resuspended with PBS and sorted by fluorescence using flow cytometry. Wild-type MSR-1 was used as a blank control. A fluorescence positivity threshold was set, and single clones with high fluorescence signals were collected and inoculated into liquid medium for expansion culture to obtain the target bifunctional engineered magnetotactic bacteria MSR-1-MagLov.

[0039] Functional validation of the engineered bacteria: Transmission electron microscopy (TEM) was performed on the engineered bacteria MSR-1-MagLov. The results showed that the intracellular magnetosomes were arranged in a regular chain-like pattern, which was not significantly different from that of wild-type MSR-1, and the magnetotactic activity retention rate was ≥92%. A gradient magnetic field of 50–800 mT was applied, and the changes in bacterial fluorescence intensity were detected. The results showed that the fluorescence intensity decreased in a gradient manner with increasing magnetic field strength, decreasing by 68% under a 500 mT magnetic field. After the magnetic field was removed, the fluorescence intensity recovered to its initial value within 30 seconds. After 50 consecutive switching of the magnetic field, the fluorescence modulation performance did not show significant attenuation, verifying that it possesses a stable magnetically controlled fluorescence switching function.

[0040] Figure 3 This is a transmission electron microscopy (TEM) image of the bifunctional engineered magnetotactic bacteria MSR-1-MagLov. The image shows the complete morphology of the engineered bacteria, with intracellular magnetosomes arranged in a regular single-stranded pattern, exhibiting intact crystal form and uniform distribution. The structure is not significantly different from that of wild-type magnetotactic bacteria, demonstrating that the expression of the fusion protein did not affect magnetosome synthesis or the cell's magnetotactic activity, thus verifying the structural integrity of the engineered bacteria.

[0041] Example 3: Co-fermentation culture of bifunctional engineered magnetotactic bacteria This embodiment describes a method for the large-scale fermentation preparation of engineered magnetotactic bacteria MSR-1-MagLov.

[0042] Seed culture preparation: The selected positive engineered bacteria were inoculated into 50 mL of MSGM seed culture medium and cultured under micro-aerobic conditions at 30℃ and 150 rpm until OD. 600 =1.0, as the primary seed culture. The primary seed culture was inoculated into 500 mL of MSGM medium at a volume fraction of 5% and cultured under the same conditions until OD... 600 =1.2, used as seed liquid for secondary fermentation.

[0043] Fermentation and Parameter Control: The secondary seed culture was inoculated into a 5 L fully automated fermenter at a volume fraction of 8%. The fermentation medium was modified MSGM medium. The fermentation conditions were controlled as follows: temperature 29℃, pH 7.0±0.1, dissolved oxygen (DO) 3%, stirring speed 150 rpm, iron source concentration of ferric ammonium citrate to a final concentration of 50 μM, and induction timing based on cell OD. 600 When the concentration of dissolved oxygen reaches 0.8, precise dissolved oxygen regulation and iron ion supplementation are initiated. The fermentation cycle is 48 hours, and samples are taken every 6 hours to detect cell concentration, magnetosome synthesis, and fluorescence expression intensity.

[0044] Fermentation results: After fermentation, the cell OD... 600 The concentration can reach 2.8, the magnetosome content is 18.5 mg / g stem cells, the bacterial cell fluorescence expression is stable, and the magnetotactic orientation rate is ≥88%, achieving optimal synergy between bacterial cell growth, magnetosome synthesis and magnetically controlled fluorescent protein expression.

[0045] Example 4: Establishment and Application of Magnetic Field Differential Fluorescence Imaging Method This embodiment establishes and verifies a magnetic field differential fluorescence imaging method based on bifunctional engineered bacteria.

[0046] Cellular imaging validation: The engineered bacterium MSR-1-MagLov was co-cultured with mouse breast cancer cells 4T1, with experimental and control groups established. In the absence of a magnetic field, a fluorescence microscope was used to acquire the first fluorescence image (Image ON), showing bright green fluorescence in the bacteria. With a magnetic field applied, a uniform 500 mT magnetic field was applied while maintaining the imaging parameters, and a second fluorescence image (Image OFF) was acquired, showing a significant decrease in bacterial fluorescence. Differential processing was performed using ImageJ software to perform pixel-level difference operations on the two images, removing cell autofluorescence and background noise from the culture medium, obtaining a high signal-to-noise ratio image that retained only the specific signal of the engineered bacteria. The results showed that the signal-to-noise ratio was improved by 12.6 times after differential processing, clearly distinguishing the adhesion and distribution of individual bacteria on the cell surface. Each group was tested in triplicate, and the results were statistically significant (P < 0.05).

[0047] In vivo animal imaging verification: SPF-grade BALB / c nude mice were selected to construct a 4T1 subcutaneous tumor model. When the tumor volume reached 100 mm³, 1×10⁻⁶ MSR-1-MagLov bacterial suspension was injected into the tumor. 8 CFU. Twenty-four hours after injection, a small animal in vivo fluorescence imaging system was used for detection. Basic fluorescence images were acquired without a magnetic field, but the fluorescence signal in the tumor area overlapped with the autofluorescence of the mouse skin, resulting in low signal discrimination. A 500 mT external magnetic field was applied to acquire low-fluorescence signal images. Differential processing was performed on the two images to obtain high signal-to-noise ratio in vivo images. Results showed that after differential processing, the colonization and distribution of engineered bacteria in the tumor tissue could be clearly observed, with an imaging depth of 6 mm. Background interference was almost completely eliminated, achieving non-invasive and precise tracing of deep tissues. This result was validated by repeated testing in three animals, and the difference in signal-to-noise ratio was statistically significant (P<0.05).

[0048] Figure 4 This is a fluorescence modulation curve of the magnetic field response of the magnetically controlled fluorescent protein MagLov. The horizontal axis represents the applied magnetic field strength in mT, and the vertical axis represents the relative fluorescence intensity. The curve shows that the fluorescence intensity of the engineered bacteria decreases in a gradient with increasing magnetic field strength, reaching the maximum decrease at 500 mT. The fluorescence recovers rapidly after the magnetic field is removed, demonstrating the reversible modulation characteristics and dose-dependent effect of the magnetically controlled fluorescence of this invention. Figure 5This is a schematic diagram of the principle and processing flow of magnetic field differential fluorescence imaging. The diagram sequentially shows: the original fluorescence image with background noise in the absence of a magnetic field, the low fluorescence image after applying a magnetic field, and the high signal-to-noise ratio specific imaging image after pixel-level differential processing. It clearly illustrates the principle, steps and noise reduction effect of the magnetic field differential imaging method of this invention. Figure 6 This image shows the effect of differential fluorescence imaging at the cellular level using a magnetic field. The image includes: A—fluorescence image of engineered bacteria co-cultured with 4T1 cells without a magnetic field; B—fluorescence image of the same field of view after applying a magnetic field; C—image after differential processing; D—bright-field control image. The results show that differential processing can eliminate interference from cellular autofluorescence. Figure 7 This image shows the results of magnetic field differential fluorescence imaging of in vivo tumor tissue. The image includes: A—raw fluorescence imaging of the tumor site in a nude mouse without a magnetic field; B—fluorescence imaging after applying a magnetic field; C—high signal-to-noise ratio imaging after differential processing; and D—in vitro fluorescence verification image of dissected tumor tissue. This demonstrates the tracing capability and imaging advantages of the method of this invention in deep in vivo tissues.

[0049] Example 5: Preparation and Tumor-Targeting Application of In-situ Magnetically Controlled Fluorescent Magnetosome Probes This embodiment provides a method for preparing magnetically controlled fluorescent magnetosome probes by in-situ lysis of engineered bacteria, and verifies their tumor cell targeting performance.

[0050] In-situ probe preparation: The engineered bacterial cells obtained from fermentation in Example 3 were collected, washed twice with PBS, and resuspended in hypotonic lysis buffer containing 1 mM EDTA and 1 mM PMSF. The cells were then sonicated in an ice bath at 200 W for 3 seconds, with 5-second intervals, for a total of 30 minutes. The lysis buffer was placed next to a permanent magnet for magnetic separation for 30 minutes. The supernatant was discarded, and the magnetically settled components were washed three times with PBS to obtain a natural phospholipid membrane-encapsulated magnetic borosiform-MagLov composite probe, which was stored at 4°C for later use.

[0051] Probe characterization: TEM characterization of the probe showed that the probe had uniform particle size, intact magnetosome crystal form, and retained biomembrane structure on the surface; fluorescence spectroscopy showed that the probe had a typical MagLov fluorescence emission peak, and the fluorescence could be reversibly regulated by a magnetic field; hysteresis loop detection showed that the probe had superparamagnetism, with a saturation magnetization of 42.6 emu / g, which can achieve rapid magnetic separation and targeted drive.

[0052] Tumor cell targeting validation: Magnetically controlled fluorescent magnetosome probes were co-cultured with 4T1 tumor cells, with a magnetic field targeting group and a non-magnetic field control group. In the magnetic field targeting group, a 300 mT magnetic field was applied next to the culture system. After 4 h of culture, magnetic field differential fluorescence imaging was used to observe probe entry into the cells. Results showed that under the influence of the magnetic field, the probes rapidly accumulated in the tumor cell region, with a cell uptake efficiency 8.3 times higher than the non-magnetic field group. The probe fluorescence signal was clear with no significant background interference, making it suitable for precise labeling and targeted tracing of tumor cells.

[0053] Figure 8 This is a characterization diagram of the in-situ magnetically controlled fluorescent magnetosome probe. The diagram includes: A—transmission electron microscopy image of the probe, showing the core and outer biomembrane structure of the magnetosome; B—fluorescence emission spectrum of the probe; C—hysteresis loop characterization diagram of the probe; D—fluorescence image of tumor cell targeting enrichment under magnetic field, comprehensively demonstrating the structure, optics, magnetism and targeting function of the probe.

[0054] Example 6: Application of engineered bacteria in whole-cell biosensors In this embodiment, bifunctional engineered bacteria are used to construct a whole-cell biosensor for heavy metal cadmium ions (Cd²⁺).

[0055] Construction of the sensing system: Utilizing the stress response characteristics of magnetotactic bacteria to heavy metal ions, the engineered bacteria MSR-1-MagLov was inoculated into MSGM medium containing different concentrations of Cd²⁺ 0, 0.1, 0.5, 1, 5, and 10 μM and cultured in a microaerobic environment for 24 h. For each concentration, three technical replicates and two biological replicates were set up.

[0056] Fluorescence signal detection and quantitative analysis: A 500 mT magnetic field was applied to each group of bacteria, and the fluorescence intensity was detected under both magnetic field and non-magnetic field conditions. The fluorescence modulation difference ΔF was calculated. Results showed a good linear relationship between Cd²⁺ concentration and the fluorescence modulation difference ΔF within the concentration range of 0.1–10 μM. The linear regression equation was Y = 12.84X + 0.21, with a linear correlation coefficient R² = 0.992. Using a signal-to-noise ratio of 3, the actual detection limit was as low as 0.08 μM, with an intra-group relative standard deviation (RSD) < 5%, representing a 2.4-fold improvement in sensitivity compared to traditional colorimetric methods. Furthermore, the magnetic field allows for rapid enrichment and separation of bacteria, avoiding interference from system impurities and significantly improving detection stability and anti-interference capabilities. This verifies that the engineered bacteria of this invention can be used for highly sensitive detection of environmental heavy metal ions, and the data exhibit good repeatability and statistical reliability.

[0057] Figure 9The response standard curve of the engineered bacterial whole-cell sensor to Cd²⁺ concentration. The horizontal axis represents the concentration of heavy metal cadmium ions in μM, and the vertical axis represents the fluorescence difference ΔF regulated by the magnetic field. The curve shows a good linear relationship in the range of 0.1–10 μM, with a linear correlation coefficient R² = 0.992, reflecting the quantitative sensing performance of the engineered bacteria in the detection of heavy metals in the environment.

[0058] Example 7 Evaluation of Tumor Targeted Therapy and Tumor Suppression Efficacy Mediated by Bifunctional Engineered Magnetotactic Bacteria SPF-grade BALB / c nude mice were selected, and 4T1 mouse breast cancer cells (5 × 10⁻⁶) were subcutaneously inoculated into the right back. 5 When the tumor volume grew to about 100 mm³, the tumors were randomly divided into three groups of 6 each: blank control group, wild-type MSR-1 group, and engineered bacteria MSR-1-MagLov combined with magnetic field targeting group.

[0059] Combined magnetic field targeted group: A permanent magnet (magnetic field strength 300 mT) was placed next to the tumor to maintain targeted enrichment for 2 h, and the engineered bacteria MSR-1-MagLov (1×10⁻⁶ mT) was injected into the tail vein. 8 CFU / animal), administered once every 3 days for a total of 4 times; the wild-type group was injected with an equal amount of wild-type MSR-1, and the control group was injected with an equal amount of sterile PBS, with all other feeding and treatment conditions being completely consistent.

[0060] The long diameter (a) and short diameter (b) of the tumor were measured every 2 days. The tumor volume was calculated using the formula V = 0.5 × a × b². The experimental period was 21 days. After the experiment, the mice were sacrificed, the tumor tissue was removed and weighed, and the tumor inhibition rate was calculated using the formula: tumor inhibition rate (%) = (1 - average tumor weight of experimental group / average tumor weight of blank control group) × 100%.

[0061] Experimental Results: In the blank control group, tumors grew rapidly, with an average tumor weight of 1.86±0.23 g after 21 days; in the wild-type MSR-1 group, the average tumor weight was 1.21±0.18 g, with a tumor inhibition rate of 34.95%; and in the engineered bacteria combined with magnetic field targeting group, the average tumor weight was only 0.62±0.11 g, with a tumor inhibition rate as high as 66.67%. Statistical analysis between groups showed that the engineered bacteria combined with magnetic field group had extremely significant differences in tumor volume and tumor weight compared with the blank control group and the wild-type group (P<0.01), proving that the engineered bacteria can significantly accumulate in tumor tissue under magnetic field targeting, effectively inhibiting tumor growth and possessing clear application value in tumor treatment.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A bifunctional engineered magnetotactic bacterium, characterized by: The engineered magnetotactic bacteria are constructed by using the recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment to construct the expression plasmid pBBR1-MagLov-MamC, and using Magnetospirillumryphiswaldense MSR-1 as the host to conjugate the recombinant expression plasmid pBBR1-MagLov-MamC, resulting in a bifunctional engineered magnetotactic bacterium MSR-1-MagLov capable of expressing the MagLov-MamC fusion protein.

2. The bifunctional engineered magnetotactic bacteria according to claim 1, characterized in that: In the recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment, MagLov, as a magnetron fluorescent protein amino acid sequence, can be replaced with either MagGFP or MagCherry.

3. The bifunctional engineered magnetotactic bacteria according to claim 1, characterized in that: In the recombinant PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment, MamC, as the amino acid sequence of the magnetosome membrane anchoring protein, can also be replaced with any one of Mms13 or MamF.

4. The bifunctional engineered magnetotactic bacteria according to claim 1, characterized in that: The engineered magnetotactic bacteria contain a recombinant expression vector with a backbone of pBBR1MCS-2 or pBBR1MCS-5. The vector contains a conjugation transfer element, an resistance selection marker, and a multiple cloning site. The resistance selection marker is a chloramphenicol resistance gene, a kanamycin resistance gene, or an ampicillin resistance gene.

5. The bifunctional engineered magnetotactic bacteria according to claim 1, characterized in that: The bifunctional engineered magnetotactic bacteria MSR-1-MagLov exhibits reversible down-regulation of fluorescence intensity by 40%-80% under a magnetic field of 50-1000 mT. The fluorescence intensity does not significantly decrease after at least 50 consecutive magnetic field switching cycles, and the exogenous gene is stably inherited without loss after at least 20 consecutive generations.

6. A method for constructing bifunctional engineered magnetotactic bacteria, characterized in that: Includes the following steps: S1. A fusion gene consisting of the wild-type MamC gene, the (Gly4Ser)3 coding sequence, and the codon-optimized MagLov gene was synthesized and inserted into an expression vector to obtain a recombinant expression vector. S2. Using an optimized conjugation transfer method, the recombinant expression vector was transformed into magnetotactic bacteria MSR-1 to obtain primary screen transformants; S3. A two-step screening method combining magnetic enrichment and fluorescence flow cytometry was used to screen for positive bifunctional engineered magnetotactic bacteria. S4. Co-fermentation culture of positive engineered bacteria to obtain engineered magnetotactic bacteria with stable magnetotactic activity and magneto-controlled fluorescence function.

7. The construction method according to claim 6, characterized in that: In step S1, the gene sequence was designed and the codons were optimized: the MagLov coding sequence was optimized using magnetotactic bacteria preferred codons, while retaining the natural sequence of the MamC gene; the Kozak ribosome binding sequence, the N-terminal signal peptide coding sequence, and the C-terminal stop codon were added, and the coding sequence of the flexible linker peptide (Gly4Ser)3 was inserted between the MamC and MagLov coding regions. EcoRI and HindIII restriction endonuclease sites were introduced at both ends of the gene, and finally the PmamC-MamC-(Gly4Ser)3-MagLov fusion gene fragment was obtained. Then, the vector was digested and ligated: the synthesized fusion gene fragment and the broad host plasmid pBBR1MCS-2 were digested with restriction endonucleases EcoR I and Hind III, respectively. The digestion products were recovered by agarose gel electrophoresis and ligated using T4 DNA ligase. Finally, transformation and positive clone screening: The ligation product was transformed into E. coli DH5α competent cells, cultured, identified by PCR, and the plasmid was extracted after amplification and culture of positive clones to obtain the recombinant expression plasmid pBBR1-MagLov-MamC.

8. The construction method according to claim 7, characterized in that: In step S2, the donor bacteria for conjugation transfer is *Escherichia coli* S17-1, with a donor-to-recipient magnetotactic bacteria ratio of 1:1 to 3:

1. The conjugation time is 4-8 hours, and the culture conditions are a microaerobic environment with an oxygen content of 1%-3% and a temperature of 28-30℃. In step S3, magnetic enrichment screening is first performed. The screened transformants are inoculated into the culture medium. After cultivation, the bacterial solution is placed next to a permanent magnet with a magnetic field strength of 300 mT to enrich magnetically sedimentation-positive strains. Then, the magnetically sedimentation-positive strains are subjected to fluorescence flow cytometry rescreening. Single clones with high fluorescence signals are collected and inoculated into liquid culture medium for expansion culture to obtain the target bifunctional engineered magnetotactic bacteria MSR-1-MagLov. Finally, the function of the engineered bacteria is verified to obtain strains that meet the requirements. In step S4, the parameters for co-fermentation culture are: dissolved oxygen 1%-5%, iron source concentration 20-80 μM, and induced OD... 600 The concentration should be 0.6-0.9, pH 6.8-7.2, and the culture temperature 28-30℃.

9. A magnetic field differential fluorescence imaging method using bifunctional engineered magnetotactic bacteria as described in any one of claims 1-5, characterized in that: Includes the following steps: (1) Under conditions without an external magnetic field, the first fluorescence image of the sample to be tested or the test subject is acquired, and the engineered bacteria are in a high fluorescence state; (2) Apply an external magnetic field of 50-1000 mT and collect the second fluorescence image. The engineered bacteria are in a low fluorescence state. (3) Perform pixel-level difference operation between the first fluorescence image and the second fluorescence image to remove background autofluorescence interference and obtain high signal-to-noise ratio target fluorescence imaging results; the imaging method is applicable to cell samples, tissue sections or live animals and can achieve non-invasive fluorescence imaging of deep tissues with a depth of not less than 5 mm.

10. Application of bifunctional engineered magnetotactic bacteria, characterized by: This includes applications in the preparation of in vivo tracer reagents, in the preparation of integrated tumor diagnostic and therapeutic agents, and in the preparation of whole-cell biosensors.

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