Marine overboard person positioning sheet and positioning system based on photobacterium

Abstract

According to the marine overboard personnel positioning sheet and positioning system based on the photogenic bacteria, escherichia coli is adopted as a chassis organism, a luxABCDEFG gene and an IPTG inducible promoter are inserted, and controllable luminescence is achieved; and culturing the strain, freeze-drying, packaging the strain, IPTG powder and nutritional ingredient powder in a 500-1000 Da dialysis bag, reviving when meeting water, and emitting light. Experimental results show that the luminescence time of the escherichia coli containing the luciferase report system gene is greater than or equal to 6 hours; the freeze-dried bacterial powder can be stored at normal temperature for more than or equal to 6 months, and the wavelength of emitted blue light is 480-520 nm, so that the problems of weak positioning signal, short duration and easiness in confusion with ambient light of the overboard personnel at night are solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of engineering bacteria, and relates to application of luminescent bacteria, in particular to a marine fallen personnel positioning sheet and a positioning system based on luminescent bacteria. BACKGROUND

[0002] Marine search and rescue of fallen personnel is a complex and challenging task, and environmental conditions, technical means and resource constraints directly affect the efficiency of marine search and rescue. The traditional search and rescue methods are as follows: (1) visual search and rescue: relying on the naked eye observation of the crew, helicopter crew or unmanned aerial vehicle operator; only suitable for daytime, good visibility, greatly limited by weather (such as fog, rain, waves) and light (night). (2) Radar search and rescue: using the radar of a ship or an airplane to detect the reflection signal of the fallen person; suitable for medium and short distance search, especially at night or in low visibility; if the fallen person does not carry metal objects or a radar reflector, the signal is very weak, and the sea wave clutter interference is serious, and small targets are easily covered. (3) Infrared / thermal imaging technology: locating the fallen person through the temperature difference between the body temperature and the sea water, but the body temperature decreases and the temperature difference decreases after the fallen person is immersed for a long time, and the equipment cost is high, which needs close-range scanning. (4) Radio positioning beacon (EPIRB / PLB), which needs the fallen person to carry an emergency beacon to emit a distress signal, and cannot be used if the fallen person does not carry the beacon, and there is a situation that the signal may be blocked by the sea waves or the battery is exhausted. Therefore, the traditional marine search and rescue relies on sound and light physical devices (such as reflective tape and position light) and electronic positioning equipment (such as AIS and infrared imaging), which have the problems of small light source, short duration and easy environmental interference.

[0003] Bioluminescence is a technology that uses chemical reactions in living organisms to produce light energy, which has made significant progress in medical diagnosis and detection, biological lighting, ecological tourism and decoration, environmental monitoring and other fields in recent years. However, existing bioluminescence technology focuses on laboratory research and lacks engineering application in marine environment. SUMMARY

[0004] The present application is aimed at the above problems, and provides a long-acting and highly visible positioning scheme by modifying luminescent bacteria through synthetic biology and combining packaging and detection technology.

[0005] The research process of the present application is as follows: first, strain modification is performed, Escherichia coli is used as a chassis organism, luxABCDEFG genes and IPTG inducible promoters are inserted to realize controllable luminescence; then the strain is cultured and freeze-dried, and it is packaged together with IPTG powder and nutrient ingredient powder in a 500-1000 Da dialysis bag, which can revive and emit light for more than 6 hours when meeting water, and the dialysis bag limits the diffusion of the bacterial body, thereby enhancing the local light intensity.

[0006] Based on the above research, the technical scheme of the present application is as follows:

[0007] In the first aspect of the present application, the application of genetically engineered luminescent bacteria in the preparation of offshore night positioning products is provided.

[0008] Preferably, the genetically engineered luminescent bacteria contain bioluminescence modules regulated by the lux operon, and the strain can be induced to emit light under rehydration conditions after freeze-drying treatment.

[0009] Further, the luminescent strain uses E. coli as the base bacteria, and its genetic circuit contains Lux ABCDEFG genes regulated by IPTG-induced promoters.

[0010] The nucleotide sequence of the plasmid gene is shown in SEQ ID NO. 1. The nucleotide sequence of the gene cluster luxC is shown in SEQ ID NO. 1 at positions 7-1449; the nucleotide sequence of the gene cluster luxD is shown in SEQ ID NO. 1 at positions 1462-2385; the nucleotide sequence of the gene cluster luxA is shown in SEQ ID NO. 1 at positions 2434-3516; the nucleotide sequence of the gene cluster luxB is shown in SEQ ID NO. 1 at positions 3531-4514; the nucleotide sequence of the gene cluster luxE is shown in SEQ ID NO. 1 at positions 4693-5805; the nucleotide sequence of the gene cluster luxG is shown in SEQ ID NO. 1 at positions 5829-6536; and the nucleotide sequence of the gene cluster luxF is shown in SEQ ID NO. 1 at positions 6552-7244.

[0011] The experimental results show that after constructing E. coli luminescent bacteria containing luciferase reporter system genes, adding inducer IPTG, and observing the luminescence, the obtained E. coli luminescent bacteria can emit obvious blue-green light in naked eye observation, and the naked eye observation luminescence time can reach about 2h, proving that the construction of E. coli luminescent bacteria containing luciferase reporter system genes is successful.

[0012] After preparing the E. coli luminescent freeze-dried bacteria, the luminescence was detected after rehydration, and the results showed that the obtained luminescent freeze-dried bacteria could emit obvious blue-green light in naked eye observation, and the naked eye observation luminescence time could reach about 4h, proving that the preparation of freeze-dried luminescent bacteria was successful and could effectively emit light.

[0013] Further, the offshore night positioning product is a sheet-shaped product with a certain area, such as 1~1.5m×1~1.5m, and the sheet-shaped large-area product can realize large-area luminescence, which is helpful for search and rescue.

[0014] The second aspect of the present application provides a luminescent bacteria-based marine personnel positioning sheet, comprising a sheet-shaped dialysis bag and genetically engineered luminescent bacteria powder, bacterial growth nutrient ingredient powder and IPTG powder arranged in the dialysis bag.

[0015] In daily life, the positioning sheet can be folded and stored in a specific pocket of clothes, such as an arm pocket or an upper pocket, so as not to affect daily life and not to make the positioning sheet particularly easy to contact water. When falling into water by accident, the positioning sheet is taken out and unfolded, seawater enters the bag through the dialysis holes of the dialysis bag, the IPTG promoter induces the expression of the Lux ABCDEFG gene, and the bacterial luminescence is realized.

[0016] Preferably, the dialysis bag is a 500-1000 Da dialysis bag for controlling the release of the bacterial strain and gathering the luminescent signal.

[0017] Further, the sheet-shaped dialysis bag is provided with a sandwich layer, and the upper and lower surfaces of the sandwich layer are distributed with mixed powder of genetically engineered luminescent bacteria powder, bacterial growth nutrient ingredient powder and IPTG powder. As for the distribution of the mixed powder on the sandwich layer, it can be evenly distributed within a certain thickness, or it can be distributed in a parabolic manner to make the luminescent intensity of the valley peak area the strongest.

[0018] Further, the thickness of the sandwich layer is 1-5 μm, and the mixed powder is adsorbed on the surface of the membrane by van der Waals force, electrostatic interaction or hydrophobic interaction. The membrane is soaked in the bacterial solution, and is placed or slightly shaken, and the adsorption efficiency is optimized by adjusting the pH, ionic strength or temperature.

[0019] The third aspect of the present application provides a marine personnel positioning system, comprising a luminescent bacteria-based marine personnel positioning sheet and a detection device. The luminescent bacteria-based marine personnel positioning sheet is as described above; the photoelectric sensor of the detection device can recognize blue light with a wavelength range of 480-520 nm, which is distinguished from the spectrum of marine natural luminous organisms.

[0020] The detection device can be carried in a drone, when it detects a blue light signal, it first converts the data through the data analysis module in it, then transmits the position and image data to the ground workstation, the workstation corrects and analyzes the image to determine whether it is a target blue light, then calculates and judges the specific position of the fallen person, and then transmits the specific position information to the drone to implement rescue.

[0021] The beneficial guarantee and effect of the present application: the luminescent duration of the E. coli containing the luciferase reporter system gene constructed by the present application is greater than or equal to 6 hours; the freeze-dried bacterial powder can be stored at room temperature for greater than or equal to 6 months, and the blue light emitted has a wavelength of 480-520 nm, thereby solving the problems of weak positioning signal, short duration and easy confusion with environmental light of a fallen person at sea at night. BRIEF DESCRIPTION OF DRAWINGS

[0022] The present disclosure is further described below in conjunction with the accompanying drawings, which are shown only for the purpose of illustrating embodiments of the present disclosure and are not intended to limit the scope of the present disclosure.

[0023] Figure 1 A gene circuit design diagram is shown;

[0024] Figure 2 A preliminary luminescence condition of the luminescent E. coli containing the luciferase reporter system gene is shown;

[0025] Figure 3 A rehydration luminescence condition of the freeze-dried E. coli luminescent bacteria in a filter membrane is shown;

[0026] Figure 4 A dialysis bag packaging system schematic diagram is shown;

[0027] Figure 5 A work flow diagram of the unmanned aerial vehicle detection system is shown.

[0028] In the figure: 1, a 500-1000 Da aperture sealed dialysis bag; 2, a modified bacterial freeze-dried powder; 3, a bacterial growth nutrient component powder; 4, an IPTG powder. DETAILED DESCRIPTION

[0029] The following examples and experimental examples further illustrate the present application and are not to be construed as limiting the same. The examples do not include detailed descriptions of conventional methods, such as PCR methods. Such methods are well known to those of ordinary skill in the art and are described in many publications.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application, and the preferred methods and materials are described below merely by way of example.

[0031] Example 1, construction of luminescent E. coli containing luciferase reporter system gene

[0032] I. Experimental materials

[0033] 1. Experimental strains and plasmids

[0034] BL21 competent cells: a tool for molecular biology experiments Escherichia coli competent cells, Beijing Quansijin Biotechnology Co., Ltd. product.

[0035] Plasmid pET28a (+): an Escherichia coli expression vector, using T7 RNA polymerase promoter, can efficiently express the target protein, Shanghai Shengong Biotechnology Service Co., Ltd. product.

[0036] 2、Experimental reagents

[0037] Table 1, the experimental reagents involved in constructing luminescent bacteria

[0038]

[0039] 3、Experimental equipment

[0040] Table 2, the experimental equipment involved in constructing luminescent bacteria

[0041]

[0042] 4、Main reagent formula

[0043] LB liquid medium: contains 10 g of tryptone, 10 g of NaCl, 5 g of yeast extract per 1 L

[0044] LB solid medium: contains 10 g of tryptone, 10 g of NaCl, 5 g of yeast extract, 15 g of agar per 1 L.

[0045] II、Experimental method

[0046] 1、Constructing plasmid containing luciferase reporter system gene

[0047] See Figure 1, construct a plasmid containing the luciferase reporter system, take pET28(+) plasmid as a skeleton, insert the gene cluster luxC, luxD, luxA, luxB, luxE, luxG, luxF containing the luciferase reporter system from 5' end to 3' end into the site between BamHI and XhoI. The nucleotide sequence of the plasmid gene is shown in SEQ ID No. 1; the nucleotide sequence of the gene cluster luxC is shown in SEQ ID No. 1 at positions 7-1449; the nucleotide sequence of the gene cluster luxD is shown in SEQ ID No. 1 at positions 1462-2385; the nucleotide sequence of the gene cluster luxA is shown in SEQ ID No. 1 at positions 2434-3516; the nucleotide sequence of the gene cluster luxB is shown in SEQ ID No. 1 at positions 3531-4514; the nucleotide sequence of the gene cluster luxE is shown in SEQ ID No. 1 at positions 4693-5805; the nucleotide sequence of the gene cluster luxG is shown in SEQ ID No. 1 at positions 5829-6536; and the nucleotide sequence of the gene cluster luxF is shown in SEQ ID No. 1 at positions 6552-7244. The plasmid gene is sent to Shanghai Sangon Biological Engineering Technology Service Co., Ltd. for synthesis.

[0048] 2. Construction of E. coli luminescent bacteria containing luciferase reporter system genes

[0049] The plasmid containing the luciferase reporter system genes is transformed into BL21 competent cells by heat shock transformation method, and is coated on a LB plate containing kanamycin. The plate is placed in an incubator at 37℃ overnight. On the second day, single colonies are picked and added to LB liquid medium containing kanamycin. The plate is placed in an incubator at 37℃ overnight at 180 rpm. On the third day, LB liquid medium containing kanamycin is added at a ratio of 1:100. When the OD value of the bacterial liquid is in the range of 0.6-0.8, an inducer IPTG is added, and the luminescence is observed.

[0050] III. Experimental results

[0051] The obtained E. coli luminescent bacteria can emit obvious blue-green light in naked eye observation. The results are shown in Table 1, and the luminescence time in naked eye observation can reach about 2 h, proving that the E. coli luminescent bacteria containing the luciferase reporter system genes are successfully constructed. Figure 2

[0052] Example 2. Preparation of E. coli luminescent freeze-dried bacteria and rehydration luminescence in filter membrane

[0053] I. Experimental materials

[0054] 1. Experimental strain

[0055] ​E. coli containing luciferase reporter system gene, prepared and preserved by Example 1.

[0056] 2. Experimental reagents

[0057] Table 3. Experimental reagents involved in the preparation of freeze-dried bacteria and the luminescence after rehydration

[0058]

[0059] 3. Experimental equipment

[0060] Table 4. Experimental equipment involved in the construction of luminescent bacteria

[0061]

[0062] 4. Main reagent formula

[0063] LB liquid medium: contains 10 g of tryptone, 10 g of NaCl, 5 g of yeast extract per 1 L;

[0064] Freeze-drying protectant: 10% skimmed milk powder + 5% trehalose, dissolved in PBS solution.

[0065] II. Experimental methods

[0066] 1. Preparation of E. coli luminescent freeze-dried bacteria

[0067] The activated E. coli luminescent bacteria were placed in a constant temperature incubation shaker at 37°C and 180 rpm for overnight culture. The next day, the precipitated bacterial cells were obtained by centrifugation. According to the concentration of the bacterial solution before freeze-drying, three groups were set up (Group A: 5 x 10 9 CFU / mL, Group B: 10 x 10 9 CFU / mL, Group C: 20 x 10 9 CFU / mL), and 2 mL of freeze-drying protectant was added to each freeze-dried bacteria. The freeze-dried bacteria were placed in a freeze dryer overnight and stored in a -20°C refrigerator.

[0068] 2. Rehydration of freeze-dried bacteria in filter membrane and observation of luminescence

[0069] One portion of freeze-dried bacteria was taken from each group, 0.1 g of IPTG and 2 mL of LB liquid medium were added, and after complete dissolution, the dialysis bag with a pore size of 500-1000 Da (purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.) was placed in pure water liquid and placed on a horizontal shaker to observe the rehydration and luminescence.

[0070] III. Experimental results

[0071] The obtained luminescent freeze-dried bacteria emitted obvious blue-green light under naked eye observation, and the results were as follows: Figure 3As shown, the luminescence duration observed by the naked eye can reach about 4 hours, proving that the freeze-dried luminescent bacteria were successfully prepared and can effectively emit light, thus having application value.

[0072] Example 3: Marine Personnel Positioning System Based on Luminescent Bacteria

[0073] See the schematic diagram of the marine underwater locator based on luminescent bacteria. Figure 4 The dialysis bag includes a sheet-like dialysis bag 1 and genetically engineered luminescent bacteria powder 2, bacterial growth nutrient powder 3, and IPTG powder 4, which are placed inside the dialysis bag. The mass ratio of the genetically engineered luminescent bacteria powder, bacterial growth nutrient powder, and IPTG powder is 1:3~5:0.01~0.1.

[0074] The dialysis bag is a 500-1000 Da dialysis bag used to control the release and aggregation of luminescent signals by the bacterial strain. To achieve a relatively uniform distribution of the powder, the dialysis bag is designed as a sheet with an internal interlayer. The upper and lower surfaces of the interlayer contain a mixture of genetically engineered luminescent bacterial powder, bacterial growth nutrient powder, and IPTG powder. Regarding the distribution of the mixed powder on the interlayer, it can be evenly distributed to a certain thickness, or it can be distributed in a parabolic pattern to maximize the luminescence intensity in the trough and peak areas.

[0075] Furthermore, the interlayer thickness is 1~5μm, and the mixed powder is adsorbed onto the membrane surface by van der Waals forces, electrostatic interactions, or hydrophobic interactions. During operation, the membrane is immersed in the bacterial solution and allowed to stand or be gently agitated. The adsorption efficiency is optimized by adjusting the pH, ionic strength, or temperature.

[0076] Under normal circumstances, the positioning tablet can be folded and stored in a specific pocket of clothing, such as an arm pocket or an upper pocket, in a way that does not interfere with daily life and prevents the tablet from easily coming into contact with water. If it accidentally falls into the water, the positioning tablet can be removed and unfolded. Seawater enters the dialysis bag through the dialysis port, and the IPTG promoter induces the expression of the Lux ABCDE gene, resulting in bacterial luminescence.

[0077] The device used in conjunction with the positioning plate is a detection device that specifically detects the blue light emitted by the bacteria. Its photoelectric sensor can identify blue light emitted by luminescent bacteria in the wavelength range of 480-520 nm, which can be distinguished from the spectrum of marine bioluminescent organisms.

[0078] The detection device is mounted inside the drone, serving as its data collection module. Figure 5The unmanned aerial vehicle detection system workflow diagram is shown: when the photoelectric sensor detects the blue light signal, the data detection module first judges the wavelength range, determines that it is a 480-520 nm wavelength range light, first processes the image through the image processing module, then transmits the position and image data to the ground workstation, the workstation corrects and analyzes the image to determine whether it is a target blue light, then calculates and judges the specific position of the fallen person, and then transmits the specific position information to the unmanned aerial vehicle to implement rescue.

[0079] The nucleotide sequence of the plasmid gene (SEQ ID NO. 1) is as follows:

[0080]

[0081] The unexplained parts involved in the present application are the same as or realized by using the prior art. The applicant declares that the present application is illustrated by the above detailed method, but the present application is not limited to the above detailed method, i.e. it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. Application of genetically engineered luminescent bacteria in preparation of marine night positioning products.

2. Use according to claim 1, characterized in that, The genetically engineered luminescent bacteria comprise a bioluminescence module regulated by a lux operon, and the bacteria can be induced to emit light under rehydration after freeze-drying.

3. Use according to claim 2, characterized in that, The luminescent bacteria strain is based on Escherichia coli, and the genetic circuit comprises Lux ABCDEFG genes regulated by a promoter induced by IPTG.

4. Use according to claim 2, characterized in that, The nucleotide sequence of the gene cluster luxC is shown in SEQ ID NO. 1 at positions 7-1449; the nucleotide sequence of the gene cluster luxD is shown in SEQ ID NO. 1 at positions 1462-2385; the nucleotide sequence of the gene cluster luxA is shown in SEQ ID NO. 1 at positions 2434-3516; the nucleotide sequence of the gene cluster luxB is shown in SEQ ID NO. 1 at positions 3531-4514; the nucleotide sequence of the gene cluster luxE is shown in SEQ ID NO. 1 at positions 4693-5805; the nucleotide sequence of the gene cluster luxG is shown in SEQ ID NO. 1 at positions 5829-6536; and the nucleotide sequence of the gene cluster luxF is shown in SEQ ID NO. 1 at positions 6552-7244.

5. The use according to claim 1, characterized in that, The marine night positioning product is a sheet-shaped product with a certain area.

6. A photobacterium-based marine man-overboard personnel positioning sheet, characterized by, The sheet-shaped dialysis bag comprises genetically engineered luminescent bacteria powder, cell growth nutrient ingredient powder, and IPTG powder arranged in the dialysis bag, The genetically engineered luminescent bacteria powder is the genetically engineered luminescent bacteria according to any one of claims 1-4. The mass ratio of the genetically engineered luminescent bacteria powder, the cell growth nutrient ingredient powder, and the IPTG powder is 1:3-5:0.01-0.

1.

7. The photobacterium-based marine man-overboard positioning sheet according to claim 6, characterized by The dialysis bag is a 500-1000 Da dialysis bag; The sheet-shaped dialysis bag is provided with a sandwich layer, and the upper and lower surfaces of the sandwich layer are uniformly distributed with mixed powder of genetically engineered luminescent bacteria powder, cell growth nutrient ingredient powder, and IPTG powder.

8. The photobacterium-based marine man-overboard positioning sheet according to claim 7, characterized by The thickness of the sandwich layer is 1-5 μm, and the mixed powder is adsorbed on the surface of the membrane by van der Waals force, electrostatic interaction, or hydrophobic interaction.

9. A marine man-overboard positioning system, characterized by The marine faller positioning sheet based on luminescent bacteria and a detection device, The marine faller positioning sheet based on luminescent bacteria is according to any one of claims 6-8; The photoelectric sensor of the detection device can recognize blue light with a wavelength of 480-520 nm.

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