FRET-based ATP gene-encoding sensor, its construction method and its application

Abstract

This invention provides a FRET-based ATP gene-encoded sensor, its construction method, and its applications. The sensor's amino acid sequence is as shown in SEQ ID NO. 6 or a variant with at least 90% sequence identity to SEQ ID NO. 6 and retaining ATP binding and FRET response functions. The sensor comprises a donor fluorescent protein cpmCherry, an ATP-binding domain, and a receptor fluorescent protein miRFP670nano3, with the structure cpmCherry-linker peptide 1-ATP-binding domain-linker peptide 2-miRFP670nano3. This invention also provides a nucleic acid molecule encoding the sensor, a recombinant vector containing the nucleic acid molecule, recombinant cells, and a method for constructing the sensor. This method includes screening FRET-adaptive fluorescent protein pairs for the ATP-binding domain, cyclically modifying mCherry, constructing a fusion protein backbone, optimizing the linker peptide, performing error-prone PCR random mutations, and high-throughput screening. This sensor can be applied to real-time dynamic monitoring of ATP in live bacteria, including monitoring dynamic changes in ATP throughout the bacterial growth cycle, detecting the effect of PCK gene overexpression on intracellular ATP levels, studying bacterial energy metabolism, or screening for enzyme-directed evolution.

Description

Technical Field

[0001] This invention relates to the field of biosensors, specifically to an adenosine triphosphate (ATP) gene-encoded sensor based on fluorescence resonance energy transfer (FRET), its construction method, and its application. Background Technology

[0002] Adenosine triphosphate (ATP) is the most important energy carrier in living organisms, participating in the regulation of almost all intracellular biochemical reactions. Changes in ATP levels are closely related to cellular metabolic state, energy balance, and various physiological and pathological processes. Therefore, real-time monitoring of intracellular ATP concentration is of great significance for studying cellular metabolism, energy regulation mechanisms, and the occurrence and development of related diseases.

[0003] Currently, methods for detecting ATP mainly include luciferase assays, high-performance liquid chromatography (HPLC), and biosensors based on fluorescence resonance energy transfer (FRET). Among these, FRET-based ATP biosensors have attracted considerable attention due to their ability to achieve real-time dynamic monitoring of ATP within living cells. FRET refers to the phenomenon where, when a donor fluorophore and a recipient fluorophore are spatially close enough, the energy of the excited donor fluorophore is transferred non-radiatively to the recipient fluorophore, causing the recipient fluorophore to emit fluorescence. Biosensors constructed based on the FRET principle typically consist of a donor fluorescent protein, a ligand-binding domain, and a recipient fluorescent protein. When the ligand-binding domain binds to the target molecule, it causes a conformational change, leading to alterations in the distance or relative orientation between the donor and recipient fluorescent proteins, thus affecting FRET efficiency. Detection of the target molecule can be achieved by detecting changes in the fluorescence signal.

[0004] In the field of biosensors, FRET technology has been widely used to construct various small molecule detection tools. For example, CN116589594A discloses a biosensor based on a truncated human sweet taste receptor extracellular domain. This sensor is a fusion protein, consisting of red fluorescent protein, linker domain 1, a truncated human sweet taste receptor T1R2 or T1R3 extracellular domain, linker domain 2, and green fluorescent protein from the N-terminus to the C-terminus, used for qualitative detection of sweet substances. CN116554350B describes a biosensor based on a human sweet taste receptor protein, in which the N-terminus of the human sweet taste receptor protein T1R3 is linked to red fluorescent protein via linker 1, and the C-terminus is linked to green fluorescent protein via linker 2, used for the detection of carbohydrates.

[0005] Regarding the selection of FRET fluorescent protein pairs, CN109762050B provides fluorescent protein pairs suitable for high-sensitivity FRET imaging, including red and green fluorescent proteins mutated at specific sites. These fluorescent proteins exhibit good photostability and can be used for high-sensitivity FRET imaging. Furthermore, CN112831482B discloses a method for rapid detection of 5-phosphoribose-1-pyrophosphate (PRPP) and the biosensor used therein. This sensor utilizes hypoxanthine-guanine phosphoribosyltransferase (HGPRT) as a ligand-binding domain and GFPmut2 fluorescent protein as a reporter gene. CN119241719A describes a glycolic acid-responsive fluorescent probe comprising an acid-sensitive peptide, a cyclically rearranged fluorescent protein, and a linker peptide, enabling rapid, real-time quantitative detection of glycolic acid.

[0006] However, existing FRET-based ATP sensors still face several technical challenges. First, current ATP sensors suffer from poor fluorescence signal stability during in vitro property monitoring, hindering long-term dynamic monitoring. Second, these sensors exhibit slow response times, poor signal-to-noise ratios, and phototoxicity, failing to capture rapid physiological fluctuations in ATP during the growth cycle of live bacteria and thus failing to meet real-time monitoring requirements. Third, existing ATP sensors are prone to folding errors, signal drift, or sluggish responses in the intracellular bacterial environment, primarily suitable for mammalian cell imaging, with few reports of long-term real-time dynamic monitoring of ATP within live bacteria. Finally, most existing ATP sensors utilize visible light-excited fluorescent proteins, which exhibit significant phototoxicity during long-term imaging, limiting their application in long-term dynamic monitoring of live cells.

[0007] Therefore, there is an urgent need to develop a novel FRET-based ATP sensor that features stable signal, rapid response, high signal-to-noise ratio, and low phototoxicity, enabling long-term real-time dynamic monitoring of ATP within living bacteria and providing a powerful tool for bacterial energy metabolism research. Summary of the Invention

[0008] To address the technical challenges of existing FRET-based ATP sensors in terms of fluorescence signal stability, response speed, bacterial intracellular applicability, and phototoxicity, and to achieve long-term, real-time dynamic monitoring of ATP within live bacteria, this invention provides a FRET-based ATP gene-encoding sensor and its construction method.

[0009] In a first aspect, the present invention provides an ATP gene encoding sensor based on FRET.

[0010] Furthermore, the ATP gene-encoded sensor comprises a donor fluorescent protein, an ATP-binding domain, and a receptor fluorescent protein; Furthermore, the donor fluorescent protein, the ATP-binding domain, and the receptor fluorescent protein are linked by a linker peptide; Furthermore, the donor fluorescent protein is an mCherry monomer or a cyclically permuted mCherry (cpmCherry), and the amino acid sequence of the mCherry monomer is SEQ ID NO.1: MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK The amino acid sequence of cpmCherry is SEQ ID NO.2: YNVDIKLDITSHNEDYTIVEQYERAEVRHSTGGMDELYKGGTGGSMVSKGVEDNMAFIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQLMYGSK AYVKHPADIPDYWKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYN; Further, the receptor fluorescent protein is miRFP670nano3, and the amino acid sequence of miRFP670nano3 is SEQ ID NO.3: MANLDKMLNTTVTEVRKFLQADRVCVFKFEEDYSGTVSHEAVDDRWISILKTQVQDRYFMETRGEEYVHGRYQAIADIYTANLVECYRDLLIEFQVRAILAVPILQGKKLWGLLVAHQLAGPREWQTWEIDFLKQQAVVMGIAIQQS; Further, the sequence of the ATP-binding domain is selected from SEQ ID NO.4 or SEQ ID NO.5, wherein SEQ ID NO.4: MKTIHVSVVTPDGPVYEDDVEMVSVKAKSGELGILPGHIPLVAPLEISAARLKKGGKTQYIAVSGGFLEVRPDNVTILAQAAERAEDIDVLRAKARKSGRTPLQSQQDDIDFKRAELALKRAMNRLSVAEMK SEQ ID NO.5: KTIHVSVVTPDGPVYEDDVEMASVKAKSGELGILPGHIPLVAPLEISAARLKKGDKTDFIAVSGGFLEVRPDKVTILAQAAERAEDIDVLRAKAAKERAERALQSQQDDIDFKAAELALKRAMNRLSVAEM Furthermore, the structure of the ATP gene-encoding sensor is cpmCherry-linker peptide 1-ATP binding domain-linker peptide 2-miRFP670nano3, and the amino acid sequence of the ATP gene-encoding sensor is SEQ ID NO.6:; In some embodiments, the ATP gene-encoded sensor is a variant that has at least 90% sequence identity with SEQ ID NO.6 and retains ATP binding and FRET response functions; Furthermore, the C-terminus of the cpmCherry is connected to the N-terminus of the ATP-binding domain via a linker peptide 1, the sequence of which is SEQ ID NO.7: GGTTTT; Furthermore, the C-terminus of the ATP-binding domain is linked to the N-terminus of miRFP670nano3 via linker peptide 2, the sequence of which is SEQ ID NO.8: TTTGAT; A second aspect of the present invention provides a nucleic acid molecule encoding the aforementioned FRET-based ATP gene encoding sensor.

[0011] A third aspect of the present invention provides a recombinant vector.

[0012] Furthermore, the recombinant vector contains the aforementioned nucleic acid molecules.

[0013] Furthermore, the recombinant vector is a prokaryotic expression vector, selected from pBAD vector or pET28a vector, preferably a pBAD vector containing an arabinose inducible promoter.

[0014] A fourth aspect of the present invention provides a recombinant cell.

[0015] Furthermore, the recombinant cells contain the aforementioned recombinant vector.

[0016] Furthermore, the recombinant cells are prokaryotic cells selected from Escherichia coli TOP10, BL21 (DE3) and / or MG1655.

[0017] A fifth aspect of the present invention provides a method for constructing the above-described ATP gene-encoding sensor.

[0018] Furthermore, the method includes the following steps: (1) Screening of FRET-compatible fluorescent protein pairs and ATP-binding domains: Based on the FPbase database and Uniprot database, fluorescent protein elements and ATP-binding domains suitable for the FRET system were screened to obtain donor fluorescent protein mCherry, acceptor fluorescent protein miRFP670nano3, and ATP-binding domain. (2) Modify the cyclic arrangement of mCherry to obtain cpmCherry: Modify the cyclic arrangement of mCherry fluorescent protein to change the dipole distance of the fluorescent protein and obtain the cyclic arrangement variant cpmCherry; (3) Construction of cpmCherry-linker peptide 1-ATP binding domain-linker peptide 2-miRFP670nano3 fusion protein backbone: cpmCherry was fused with ATP binding domain and receptor fluorescent protein miRFP670nano3 to construct the fusion protein backbone; (4) Optimize the linking peptide using NNK degenerate primers: Direct evolution of the linking peptide between fluorescent protein and ATP binding domain is performed using NNK degenerate primers, and the length and amino acid composition of the linking peptide are optimized by saturation mutation. (5) Error-prone PCR random mutation and high-throughput screening: Error-prone DNA polymerase was used to randomly mutate the fusion protein to construct a mutant library. Positive clones with significant changes in fluorescence ratio and stable signal were obtained through high-throughput screening. (6) Sequencing identified the ATP gene-encoded sensor.

[0019] A sixth aspect of the present invention provides the application of the above-described ATP gene-encoding sensor in real-time dynamic monitoring of ATP in live bacteria.

[0020] Furthermore, the applications include monitoring the dynamic changes of ATP throughout the bacterial growth cycle, detecting the effect of PCK gene overexpression on intracellular ATP levels in bacteria, and conducting bacterial energy metabolism studies or enzyme-directed evolution screening.

[0021] Furthermore, the structure of the sensor is: cpmCherry-linker peptide 1-ATP binding domain-linker peptide 2-miRFP670nano3.

[0022] The beneficial effects of this invention are as follows: (1) Excellent in vitro robustness: Through multiple rounds of engineering modification, the ATP gene-encoding sensor maintains a stable response signal in the temperature range of 25-37℃ and the pH range of 7.0-8.0, and has high selectivity for ATP (response to other nucleotides is less than 10%), which solves the problem of existing ATP sensors being prone to folding errors and signal drift in bacterial cells; (2) Real-time ATP monitoring of the entire growth cycle of live bacteria was realized for the first time: It broke through the limitation that the existing ATP sensors are only suitable for mammalian cell imaging, and realized continuous, real-time and non-destructive monitoring of ATP levels throughout the entire growth cycle of live bacteria. The fit with the detection results of commercial luciferase method reached 0.95. (3) Low phototoxicity: Near-infrared fluorescent protein miRFP670nano3 is used as the acceptor, which significantly reduces the phototoxicity during long-term imaging compared with existing visible light-excited fluorescent proteins. (4) High affinity and specificity: The Kd value is about 2 μM, indicating that it has high affinity and its specific response to ATP is significantly higher than that of other nucleotides and derivatives. Attached Figure Description

[0023] Figure 1 Figure 1A shows the structural schematic and characteristic analysis of the FRET-based ATP gene-encoded sensor. The red, pink, yellow, and gold portions represent the ATP gene-encoded sensor, respectively. Figure 1B shows the concentration response curves of the ATP gene-encoded sensor to ATP and ADP (the horizontal axis represents ATP / ADP concentration, and the vertical axis represents the fluorescence ratio). Figure 1C shows the ATP affinity fitting curve of the ATP gene-encoded sensor, with the calculated apparent dissociation constant Kd≈2 μM. Figure 1D shows the specific response histogram of the ATP gene-encoded sensor to substrates such as ATP, ADP, AMP, CTP, TTP, GTP, and UTP. Figure 1E shows the ATP response amplitude curves of the ATP gene-encoded sensor under different pH conditions (pH 4.5–8.5). Figure 1F shows the ATP response comparison curves of the ATP gene-encoded sensor at 25℃ and 37℃. Figure 1G shows the ATP binding kinetics fitting curves of the ATP gene-encoded sensor (binding rate constant Kon and dissociation rate constant Koff).

[0024] Figure 2 This is a comparative analysis chart showing the application of ATP gene-encoding sensors in monitoring dynamic changes in ATP. Figure 2 A: Comparison of the ATP concentration versus time curve during bacterial growth detected by commercial luciferase assay and the fluorescence ratio (670 nm / 603 nm) versus time curve of the ATP gene-encoded sensor; Figure 2B: Correlation fitting plot between the ATP gene-encoded sensor detection results and the luciferase assay results (R... 2 =0.94904). Figure 3 illustrates the performance comparison of the ATP gene-encoded sensor with other ATP sensors in living cells. Figure 3A shows the ATP gene-encoded sensor detecting dynamic changes in ATP during the top 10 growth cycles of bacteria; Figure 3B shows the Ateam 3.01 sensor detecting dynamic changes in ATP during the top 10 growth cycles of bacteria; Figure 3C shows the ATP gene-encoded sensor detecting dynamic changes in ATP during the growth cycle of bacteria MG1655; Figure 3D shows the mCherry-iATPsnFR1.1 sensor detecting dynamic changes in ATP during the growth cycle of bacteria MG1655; Figure 3E compares the signal-to-noise ratio of the ATP gene-encoded sensor and mCherry-iATPsnFR1.1 in detecting dynamic changes in ATP during bacterial growth cycles. Figure 4 is a comparison of the growth curves of wild-type Escherichia coli and strains expressing different ATP sensors; Figure 5 illustrates the application of the ATP gene-encoded sensor in carbon source metabolism research, specifically the changes in ATP levels detected by the ATP gene-encoded sensor in E. coli overexpressing the PCK gene. Figures 5A and 5B are shown in Figure 5A. Figure 5 D: Bacterial growth curves (OD600) of the PCK overexpression group and the control group under different conditions (no additional carbon source, 5 mM glucose, 5 mM glycerol); Figure 5B and Figure 5 E: ATP fluorescence ratio (670 nm / 603 nm) changes in the PCK overexpression group and control group under different conditions (no additional carbon source, 5 mM glucose, 5 mM glycerol); Figure 5C and Figure 5 F: EGFP expression levels in the PCK overexpression group and the control group under different conditions (no additional carbon source, 5 mM glucose, 5 mM glycerol). Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0026] Example 1 This embodiment provides a method for designing and screening ATP gene-encoding sensors, the specific process of which is as follows: 1. Screening of fluorescent protein pairs To achieve efficient fluorescence resonance energy transfer (FRET), suitable donor-acceptor fluorescent protein pairs need to be screened. Using the FRET calculate tool in the Fpbase database, a donor-acceptor distance (R0) of 6-7 nm at 50% FRET efficiency was used as the screening criterion to evaluate various fluorescent protein pairs. The final selected combination was: cyclically arranged mcherry (cpmcherry) as the donor fluorescent protein and miRFP670nano3 as the acceptor fluorescent protein.

[0027] 2. Screening of ATP-binding domains To achieve specific recognition of ATP, suitable ATP-binding domains need to be screened. Using the amino acid sequence of the reported ATP gene encoding the ATP-binding domain (amino acid sequence such as SEQ ID NO.4) as a template, homology matching was performed using the BLAST tool in the UniProt database. A series of candidate sensing domains from different sources were obtained and constructed between cpmCherry and miRFP670nano3 using molecular biology methods to create a fusion protein, which was then functionally screened. Finally, a fusion protein derived from... Bacillus sp The ATP-binding domain mutant of (strain PS3) is used as the binding domain of this sensor. Its UniProt ID is P07678 and its amino acid sequence is shown in SEQ ID NO.5.

[0028] 3. Random mutation and high-throughput screening of linker peptides and sensing domains To optimize sensor performance, random mutations and high-throughput screening were performed on linker peptide 1, linker peptide 2, and the ATP-binding domain.

[0029] Mutant library construction: Error-prone DNA polymerase (EPPCR) was used to randomly mutate the coding sequences of the linker peptide and the sensor domain, and PaqCI type II restriction endonuclease technology was used to efficiently clone the mutated fragments into the expression vector to construct the sensor mutant library.

[0030] The specific steps for high-throughput screening are as follows: (1) Single-clone culture: Select single-clone colonies from the mutant library and inoculate them into 96-well plates containing culture medium, one colony per well, and culture overnight.

[0031] (2) Protein expression: Add an inducer (such as 0.002% arabinose) to each well to induce sensor protein expression.

[0032] (3) Cell lysis: After the culture is completed, add 300 μL of lysis buffer to each well, let stand for 15 minutes to lyse the bacteria, and then centrifuge to collect the supernatant.

[0033] (4) Fluorescence detection: The fluorescence of the supernatant was detected using a microplate reader. The detection conditions were: excitation wavelength 540 nm, and simultaneous detection of emission intensity at 603 nm (donor, cpmCherry) and 670 nm (acceptor, miRFP670nano3). The fluorescence ratio (F) was calculated. 670 / F 603 Clones with high and stable ratios are potential performance-enhancing mutants.

[0034] The positive clones obtained from the initial screening were sequenced and identified. Finally, the ATP gene-encoded sensor containing the ATP binding domain mutant with the best performance was obtained and named IGAS 1.0. Its amino acid sequence is shown in SEQ ID NO.6.

[0035] Example 2 Based on Example 1, this example provides the construction, isolation, and purification process of the high-affinity ATP gene-encoded sensor IGAS 1.0 (hereinafter referred to as IGAS 1.0), specifically including the heterologous expression and purification method of IGAS 1.0 (amino acid sequence as shown in SEQ ID NO. 6) in Escherichia coli: 1. Construction of the expression carrier The nucleotide sequence encoding IGAS 1.0 was cloned into the pBAD expression vector to obtain the recombinant plasmid pBAD-IGAS1.0. The recombinant plasmid contains an arabinose-inducible promoter and an ampicillin resistance gene.

[0036] 2. Transformation of recombinant Escherichia coli The obtained recombinant plasmid pBAD-IGAS 1.0 was transformed into E. coli TOP10 competent cells. The specific steps are as follows: Take 100 μL of competent cells, add 1 μL of plasmid DNA (about 50-100 ng), and incubate on ice for 30 min; then heat shock in a 42℃ water bath for 90 s, and immediately cool on ice for 5 min; add 500 μL of antibiotic-free LB liquid medium, and revive and culture at 37℃ and 220 rpm for 1 h; take an appropriate amount of bacterial culture and spread it on LB solid medium plates containing 100 μg / mL ampicillin (formulation: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar), and incubate upside down at 37℃ for 16 h until clearly visible single colonies appear.

[0037] 3. Induced expression Single colonies obtained in step 2 were picked and inoculated into 10 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured overnight at 37°C and 220 rpm. The next day, the overnight culture was transferred to 500 mL of LB liquid medium containing the same antibiotic at a 1:100 volume ratio and cultured at 37°C and 220 rpm. When the OD600 value of the bacterial culture reached 1.0-2.0, the culture was transferred to 4°C and incubated for 30 min. Then, L-arabinose was added to a final concentration of 0.002% (w / v), and expression was induced at 37°C and 220 rpm for 5 h. The temperature of the shaker was then adjusted to 25°C, and expression was induced for another 18 h. After induction, the bacterial cells were collected by centrifugation at 5000 rpm for 20 min. The bacterial cells can be used directly for subsequent purification or stored at -20°C for later use.

[0038] 4. Protein purification 4.1 Buffer Preparation Lysis buffer: 20 mM Tris-HCl, 500 mM NaCl, add 1:10 hammer lysis buffer according to the instructions, pH 8.0.

[0039] Washing buffer: 20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0.

[0040] Elution buffer: 20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8.0.

[0041] 4.2 Cell lysis The bacterial cells collected in step 3 were resuspended in lysis buffer and placed in an ice-water bath for ultrasonic disruption. The disruption conditions were: 200 W power, 3 s operation time, 5 s interval, and a total disruption time of 30 min.

[0042] 4.3 Affinity chromatography purification The disrupted bacterial culture was centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was collected and filtered through a 0.22 μm filter membrane. A Ni-NTA gravity column was taken, washed with 5 column volumes of ultrapure water, and then equilibrated with 3 column volumes of lysis buffer. The filtered supernatant was incubated with Ni-NTA packing material at 4 °C for 1 h, and the flow-through was collected. The packing material was washed with washing buffer until no color change was observed when the flow-through was added to G250 staining solution. 5 mL of elution buffer was added, and the mixture was incubated for 5 min. The eluent was collected, which was the purified IGAS 1.0 protein.

[0043] 4.4 Purity Identification and Quantification The purified protein was analyzed by spectral analysis using a microplate reader. The presence of distinct double peaks at 603 nm and 670 nm indicated successful purification of the IGAS 1.0 protein. The protein concentration was then determined using the BCA method to quantify the IGAS 1.0.

[0044] The results are as follows Figure 1 As shown in Figure B, the high-purity ATP gene-encoding sensor IGAS1.0 was obtained using the method described above.

[0045] Example 3 Based on Examples 1 and 2, this example provides an in vitro performance characterization method for the above-mentioned ATP gene encoding sensor IGAS 1.0 (hereinafter referred to as IGAS 1.0).

[0046] 1. Preparation of protein samples The purified IGAS 1.0 protein was obtained according to the method described in Example 1 and diluted to a suitable concentration (preferably 5 μM) with assay buffer (20 mM Tris, 500 mM NaCl, pH 8.0) for subsequent characterization experiments.

[0047] 2. Affinity test Weigh ATP powder and dissolve it in 10×ATP buffer (500 mM MOPS, 20 mM MgCl2, 200 mM KCl, 0.01% Triton X-100, pH 7.2) to prepare an ATP stock solution. Dilute the ATP stock solution to a series of concentrations using assay buffer: 10 mM, 5 mM, 1 mM, 500 μM, 200 μM, 50 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM, and 0.01 μM.

[0048] 20 μL of ATP solutions of different concentrations were mixed with 180 μL of sensor protein solution in a 96-well plate. After shaking for 2 min, the fluorescence spectrum was detected using a microplate reader. The detection conditions were: excitation wavelength 540 nm, emission wavelength scan range 580-700 nm, and fluorescence intensities at 603 nm (cpm Cherry emission peak) and 670 nm (miRFP670nano3 emission peak) were recorded. The fluorescence ratio (F) at different ATP concentrations was calculated. 670 / F 603 ), plot the dose-response curve, and fit it to obtain the apparent dissociation constant (K). d，app The result is as follows: Figure 1 As shown in Figure C, the Kd value of IGAS 1.0 for ATP is approximately 2 μM, indicating that it has high affinity.

[0049] 3. Specificity assay Based on the molecular weight of each substrate, prepare substrate solutions with a final concentration of 1000 μM for the following substrates: ADP, AMP, CTP, TTP, GTP, UTP, dATP, dCTP, dTTP, and adenosine. Mix 20 μL of each substrate solution with 180 μL of sensor protein solution, and detect changes in fluorescence ratios as described in step 2. Calculate the relative response amplitudes of the other substrates, taking the response amplitude under ATP stimulation as 100%. The results are as follows... Figure 1 As shown in Figure D, IGAS 1.0's response to ATP is significantly higher than that of other nucleotides and derivatives, indicating that it has good ATP specificity.

[0050] 4. Temperature sensitivity measurement The response amplitude of IGAS 1.0 to 100 μM ATP was measured at different temperatures (25℃ and 37℃) according to the method in step 2.2. The results are as follows. Figure 1 As shown in Figure F, IGAS 1.0 maintains a stable response at different temperatures, indicating that it has good temperature tolerance.

[0051] 5. pH sensitivity measurement Prepare Tris-HCl buffer solutions with a pH range of 4.8–8.3. Dilute the sensor protein with buffers of different pH values, and determine the response amplitude to 100 μM ATP according to the method in step 2.2. The results are as follows: Figure 1 As shown in E, IGAS 1.0 exhibits the best response in the pH range of 7.0–8.0.

[0052] In summary, the results of this embodiment demonstrate that the IGAS 1.0 sensor obtained by this invention has high affinity, high specificity, and good environmental stability, exhibiting strong robustness.

[0053] Example 4 This embodiment illustrates the application of an ATP gene-encoded sensor in real-time dynamic monitoring of ATP in live bacteria. Specifically, the IGAS 1.0 prepared in the above embodiment is used to monitor the dynamic changes of ATP in real time during the growth of live bacteria.

[0054] 1. Preparation of recombinant bacteria The recombinant plasmid pBAD-IGAS 1.0 constructed in Example 1 was transformed into Escherichia coli TOP10 / BL21 to obtain a recombinant strain that stably expresses IGAS 1.0.

[0055] 2. Bacterial culture and induction Single colonies of the recombinant strain were picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin, and incubated overnight at 37°C and 220 rpm. The next day, the colonies were transferred 1:100 to fresh medium and incubated at 37°C until OD500 was reached. 600 ≈0.5. Dilute the bacterial culture to OD600=0.1, and repeat this step 5 times to ensure that the bacteria are in a stable growth state.

[0056] 3. Real-time fluorescence monitoring The treated bacterial culture was transferred to 96-well plates, 200 μL per well. Real-time monitoring was performed using a microplate reader under the following conditions: excitation wavelength 540 nm, emission wavelengths 603 nm and 670 nm, measured every 5 minutes for 18 hours. OD was also monitored. 600 The value is used to track the bacterial growth curve.

[0057] 4. Detection of dynamic changes in ATP under different conditions This embodiment sets the following three detection conditions: (1) Comparison with commercial luciferase method: Using the sensor of the present invention and the commercial luciferase ATP detection kit respectively, the changes in ATP levels during bacterial growth under the same culture conditions were monitored. The results showed that the sensor of the present invention can achieve continuous and non-destructive real-time monitoring, while the luciferase method requires cell destruction for sampling and cannot achieve dynamic tracking. Figure 2 ).

[0058] (2) Differences in real-time detection in bacteria by different ATP gene-encoded sensors ( Figure 3 ).

[0059] (3) The effect of different ATP gene-encoded sensor expression in bacteria on Escherichia coli ( Figure 4 ).

[0060] (4) Effect of PCK gene overexpression on ATP levels: The PCK gene overexpression plasmid was co-transformed with IGAS 1.0 into E. coli, and the effect of PCK overexpression on intracellular ATP levels was monitored. The results showed that PCK overexpression led to a significant increase in ATP levels. Figure 5 ).

[0061] The results are as follows Figure 3 and Figure 4 As shown, the IGAS 1.0 sensor provided by this invention can realize real-time, dynamic, and non-destructive monitoring of ATP in live bacteria, providing a powerful tool for bacterial energy metabolism research.

[0062] This embodiment demonstrates that the high-affinity ATP gene-encoding sensor IGAS 1.0 provided by the present invention can monitor the dynamic changes of ATP in live bacteria in real time.

[0063] Example 5 This embodiment provides a nucleic acid molecule encoding an ATP gene-encoded sensor. The sensor encoded by this nucleic acid molecule is a FRET-based ATP gene-encoded sensor, and its amino acid sequence is shown in SEQ ID NO. 6.

[0064] As described in Examples 1-4, the ATP gene-encoding sensor is an ATP sensor based on the fluorescence resonance energy transfer (FRET) principle, comprising three main functional modules: a donor fluorescent protein, an ATP-binding domain, and a receptor fluorescent protein. The structure of this sensor is: cpmCherry-linker peptide 1-ATP-binding domain-linker peptide 2-miRFP670nano3.

[0065] The donor fluorescent protein is a circularly arranged mCherry, i.e., cpmCherry, with an amino acid sequence as shown in SEQ ID NO.2; the amino acid sequence of the ATP-binding domain is shown in SEQ ID NO.4 or SEQ ID NO.5, and this binding domain is derived from Bacillus sp. (strain PS3), with UniProt ID P07678; the receptor fluorescent protein is miRFP670nano3, with an amino acid sequence as shown in SEQ ID NO.3.

[0066] The nucleic acid molecule in this embodiment was constructed using standard molecular cloning techniques. First, a corresponding nucleic acid coding sequence was designed based on the amino acid sequence of the ATP gene-encoding sensor (SEQ ID NO. 6), and optimized considering the codon bias of the E. coli expression system. The optimized nucleic acid sequence was synthesized and the target fragment was obtained by PCR amplification. Subsequently, this fragment was cloned into the pBAD expression vector by restriction endonuclease digestion and ligation, constructing the recombinant plasmid pBAD-ATP gene-encoding sensor.

[0067] After successful transformation into *E. coli*, this nucleic acid molecule was able to efficiently express the ATP gene-encoded sensor protein under arabinose induction, such as... Figure 1 As shown in Figure A, the expressed sensor protein retains complete ATP binding and FRET response functions, such as... Figure 1 As shown in B-1G, it can specifically recognize ATP and produce changes in fluorescence signals.

[0068] like Figure 2 As shown, the ATP gene-encoded sensor encoded by this nucleic acid molecule can monitor the dynamic changes of ATP in live bacteria in real time, which has the advantage of non-destructive detection compared with the traditional luciferase method. Figure 3The performance of the ATP gene-encoding sensor compared with other ATP sensors in living cells was demonstrated, proving that it has a higher signal-to-noise ratio. Figure 4 The growth curves of the strains expressing the ATP gene-encoded sensor were similar to those of the wild-type strains, indicating that the sensor had little impact on the growth of the host bacteria. Figure 5 This further demonstrates the application of the ATP gene-encoded sensor in carbon source metabolism research, proving that the sensor encoded by this nucleic acid molecule has broad application prospects.

[0069] Example 6 This embodiment provides a recombinant vector containing nucleic acid molecules encoding an ATP gene-encoding sensor.

[0070] As described in Example 5, the sensor encoded by this nucleic acid molecule is an ATP gene-encoded sensor based on FRET, and its amino acid sequence is shown in SEQ ID NO. 6. The ATP gene-encoded sensor contains three main functional modules: a donor fluorescent protein, an ATP-binding domain, and a receptor fluorescent protein, with the structure cpmCherry-linker peptide 1-ATP-binding domain-linker peptide 2-miRFP670nano3.

[0071] In this embodiment, the recombinant vector is a prokaryotic expression vector, selected from the pBAD series vectors or the pET28a series vectors. In a preferred embodiment, the vector is a pBAD vector containing an arabinose inducible promoter.

[0072] Specifically, the nucleotide sequence encoding the ATP gene-encoding sensor was inserted into the pBAD expression vector using molecular cloning technology to construct the recombinant plasmid pBAD-ATP gene-encoding sensor. This recombinant vector contains an arabinose-inducible promoter, allowing the expression of the ATP gene-encoding sensor to be precisely regulated by arabinose concentration. Figure 1 As shown in Figure A, after transformation into E. coli, this vector can efficiently express the ATP gene encoding a sensor protein under arabinose induction.

[0073] The sensor protein expressed by this recombinant vector retains its complete ATP binding and FRET response functions, such as... Figure 1 As shown in B-1G, it can specifically recognize ATP and produce changes in fluorescence signals. Figure 1 B shows the fluorescence emission spectra of ATP in both the free and bound states. Figure 1 C shows the dose-response curves for ATP and ADP. Figure 1 D indicates that the ATP gene-encoded sensor is specific to ATP. Figure 1 E shows the effect of pH on the fluorescence ratio of the ATP gene-encoded sensor. Figure 1F shows the response of the ATP gene-encoded sensor to ATP at different temperatures. Figure 1 G represents the kinetic parameters analysis of the ATP gene-encoded sensor.

[0074] like Figure 2 As shown, the ATP gene-encoded sensor expressed by this recombinant vector can monitor the dynamic changes of ATP in live bacteria in real time, which has the advantage of non-destructive detection compared with the traditional luciferase method. Figure 2 A compared the curves of ATP changes over time detected by the luciferase method and the ATP gene-encoded sensor. Figure 2 B shows the correlation analysis of the detection results of the two methods.

[0075] Figure 3 The performance of the ATP gene-encoding sensor compared with other ATP sensors in living cells was demonstrated, proving that it has a higher signal-to-noise ratio. Figure 3 A and Figure 3 C demonstrates how an ATP gene-encoded sensor detects changes in intracellular ATP concentration during bacterial growth. Figure 3 B showcases the performance of the Ateam 3.01 sensor. Figure 3 D shows the performance of the mCherry-iATPsnFR1.1 sensor. Figure 3 E represents the statistical analysis and comparison of signal-to-noise ratio.

[0076] like Figure 4 As shown, the growth curves of the strain expressing the ATP gene encoding the sensor are similar to those of the wild-type strain, indicating that the sensor has little impact on the growth of the host bacteria. Figure 4 The growth curves of wild-type Escherichia coli and strains expressing different ATP sensors are shown.

[0077] Figure 5 This further demonstrates the application of the ATP gene-encoded sensor in carbon source metabolism research, proving that the sensor expressed by this recombinant vector has broad application prospects. Figure 5 A and Figure 5 D shows the growth curves of the strain under different carbon source conditions. Figure 5 B and Figure 5 E demonstrates how an ATP gene-encoded sensor detects changes in intracellular ATP levels under different carbon source conditions. Figure 5 C and Figure 5 F is the curve showing the change in EGFP fluorescence intensity over time, used to monitor cell growth status.

[0078] Example 7 This embodiment provides a recombinant cell containing the recombinant vector pBAD-ATP gene-encoding sensor as described in Example 3.

[0079] The recombinant cells were prokaryotic cells selected from Escherichia coli TOP10, BL21(DE3), or MG1655. In this embodiment, Escherichia coli TOP10 was preferably used as the host cell.

[0080] The recombinant cells were prepared as follows: The pBAD-ATP gene-encoding sensor recombinant vector constructed in the above example was transformed into E. coli TOP10 competent cells. The specific transformation steps were as follows: 100 μL of competent cells were taken, 1-2 μL of plasmid DNA (50 ng) was added, and the cells were placed on ice for 30 minutes; then, the cells were heat-shocked in a 42℃ water bath for 90 seconds, and immediately placed on ice for 5 minutes; 500 μL of antibiotic-free LB liquid medium was added, and the cells were revived and cultured at 37℃ and 220 rpm for 1 hour; an appropriate amount of bacterial culture was spread on an LB solid medium plate containing 100 μg / mL ampicillin, and incubated upside down at 37℃ for 12-16 hours until clearly visible single colonies appeared.

[0081] like Figure 1 As shown in Figure A, the recombinant cells can efficiently express the ATP gene encoding a sensor protein under arabinose induction. The expressed sensor protein retains complete ATP binding and FRET response functions, and can specifically recognize ATP and generate fluorescence signal changes.

[0082] In a preferred embodiment, the recombinant cells can also be selected from Escherichia coli BL21(DE3) as the host cell. In this case, pET28a series vectors can be used as expression vectors to induce the expression of the ATP gene encoding the sensor protein via IPTG.

[0083] In another preferred embodiment, the recombinant cells may also be selected from Escherichia coli MG1655 as the host cell. This strain is more suitable for metabolic research as a model organism.

[0084] like Figure 2 As shown, this recombinant cell-expressed ATP gene-encoded sensor can monitor dynamic changes in ATP in live bacteria in real time, offering the advantage of non-destructive detection compared to the traditional luciferase method. Figure 2 A compared the curves of ATP changes over time detected by the luciferase method and the ATP gene-encoded sensor. Figure 2 B shows the correlation analysis of the detection results of the two methods.

[0085] like Figure 3 As shown, recombinant cells expressing the ATP gene encoding a sensor have a higher signal-to-noise ratio compared to cells expressing other ATP sensors. Figure 3 A and Figure 3 C demonstrates how an ATP gene-encoded sensor detects changes in intracellular ATP concentration during bacterial growth. Figure 3E represents the statistical analysis and comparison of signal-to-noise ratio.

[0086] like Figure 4 As shown, the growth curves of the recombinant cells expressing the ATP gene encoding the sensor are similar to those of the wild-type strain, indicating that the sensor has little impact on the growth of the host bacteria, thus demonstrating that the recombinant cells have good biocompatibility.

[0087] like Figure 5 As shown, recombinant cells expressing the ATP gene encoding a sensor can be applied to carbon source metabolism research, enabling real-time monitoring of dynamic changes in intracellular ATP levels under different carbon source conditions. Figure 5 B and Figure 5 E demonstrates how an ATP gene-encoded sensor detects changes in intracellular ATP levels under different carbon source conditions, proving the application value of this recombinant cell in metabolic research.

[0088] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. An ATP gene-encoding sensor based on FRET, characterized in that, The ATP gene-encoded sensor comprises a donor fluorescent protein, an ATP-binding domain, and a receptor fluorescent protein; the donor fluorescent protein, the ATP-binding domain, and the receptor fluorescent protein are linked by a linker peptide; the donor fluorescent protein is an mCherry monomer or a cyclically permuted mCherry (i.e., cpmCherry), the amino acid sequence of the mCherry monomer is shown in SEQ ID NO.1, and the amino acid sequence of the cpmCherry is shown in SEQ ID NO.

2.

2. The ATP gene-encoding sensor according to claim 1, characterized in that, The receptor fluorescent protein is miRFP670nano3, and the amino acid sequence of miRFP670nano3 is shown in SEQ ID NO.3; the sequence of the ATP binding domain is selected from SEQ ID NO.4 or SEQ ID NO.

5.

3. The ATP gene-encoding sensor according to claim 1, characterized in that, The ATP gene-encoded sensor contains cpmCherry, with the structure cpmCherry-linker peptide 1-ATP binding domain-linker peptide 2-miRFP670nano3, and the amino acid sequence of the ATP gene-encoded sensor is shown in SEQ ID NO. 6; or the ATP gene-encoded sensor is a variant that has at least 90% sequence identity with SEQ ID NO. 6 and retains ATP binding and FRET response functions.

4. The ATP gene-encoding sensor according to claim 3, characterized in that, The sequence of linker peptide 1 is shown in SEQ ID NO.7, and the sequence of linker peptide 2 is shown in SEQ ID NO.

8.

5. A nucleic acid molecule encoding the ATP gene-encoding sensor as described in any one of claims 1-4.

6. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 5, wherein the recombinant vector is a prokaryotic expression vector selected from pBAD vector or pET28a vector, preferably a pBAD vector containing an arabinose inducible promoter.

7. A recombinant cell, characterized in that, The recombinant cells comprise the recombinant vector of claim 6, wherein the recombinant cells are prokaryotic cells selected from Escherichia coli TOP10, BL21 (DE3) or MG1655.

8. The method for constructing the ATP gene-encoding sensor according to any one of claims 1-4, characterized in that, The method includes the following steps: (1) screening FRET-adaptor fluorescent protein pairs for ATP binding domains; (2) performing cyclic arrangement modification on mCherry to obtain cpmCherry; (3) constructing a cpmCherry-linker peptide 1-ATP binding domain-linker peptide 2-miRFP670nano3 fusion protein backbone; (4) optimizing the linker peptides using NNK degenerate primers; (5) performing error-prone PCR random mutation and high-throughput screening to obtain positive clones with stable signals and significant ratio changes; (6) sequencing to identify the ATP gene-encoded sensor.

9. The application of the ATP gene-encoding sensor as described in any one of claims 1-4 in real-time dynamic monitoring of ATP in live bacteria.

10. The application according to claim 9, characterized in that, The applications include monitoring the dynamic changes of ATP throughout the bacterial growth cycle, detecting the effect of PCK gene overexpression on intracellular ATP levels in bacteria, and conducting bacterial energy metabolism studies or enzyme-directed evolution screening.

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