Construction method of interactive stable expression group response mixed bacterium induction system and application in halomonas

Abstract

The invention discloses a construction method of an interactive stable expression group response mixed bacterium induction system and application in halomonas. The interactive stable expression group response mixed bacterium induction system comprises a Plux-luxR-cinI induced expression system and a Pcin-cinR-luxI induced expression system, and a wild type promoter Plux and a wild type promoter Pcin are subjected to random library building on the basis of the wild type promoter Plux and the wild type promoter Pcin, so that mutant promoters with different activity intensities are obtained; on the basis of the interactive stable expression group response mixed bacterium induction system, a target gene expression amplification system and a target gene expression inhibition system are constructed. According to the system, the cell density is used as a response signal to dynamically regulate and control a target gene, induction is realized in a mixed bacteria manner, an inducer does not need to be added externally, the actual production cost is reduced, and the application prospect of a halomonas cell factory is expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology and metabolic engineering, and in particular to a method for constructing an interactive stable expression population response mixed bacteria induction system and its application in Halomonas. Background Art

[0002] The traditional static regulation method is a metabolic engineering strategy that statically optimizes the target metabolic pathway by designing and modifying genetic coding components. It is directional and irreversible. The modified cells do not have the ability to sense the specified signal and make specific adjustments during the growth process. In addition, the static regulation of metabolic pathways may affect the growth state of cells, which to a certain extent limits the diversity of products produced by metabolic engineering microorganisms. Compared with traditional static regulation, the interactive stable expression population response mixed bacteria induction system uses cell density as a response signal, can sense the cell state online and perform real-time metabolic regulation. Therefore, the dynamic regulation and modification of metabolic pathways is a metabolic engineering strategy with great application prospects. The interactive stable expression population response mixed bacteria induction system uses cell density as a response signal, can dynamically regulate metabolic pathways, decouple the cell growth of the engineered strain from the product production process, reduce the burden of product production on cell growth and the adverse reactions of the product to the cell, and the induction system does not require exogenous addition of inducers. In actual production, it can effectively reduce the high cost of using inducers, simplify downstream processing, and improve production efficiency.

[0003] Compared with traditional metabolic engineering chassis bacteria, the present invention focuses on microorganisms that survive in extreme environments, especially Halomonas that can perform sterilization-free open fermentation. Compared with exogenous addition of inducers, the interactive and stable expression of the quorum response mixed bacteria induction system in Halomonas can be induced by mixed bacteria to achieve dynamic regulation of metabolic pathways, and no bacterial contamination will occur. However, static regulation and transformation of metabolic pathways is currently one of the most commonly used metabolic engineering transformation methods for fermentation production using Halomonas as chassis cells. Due to its directional, irreversible and potential adverse effects of products on cells, the diversity of product types is limited, limiting the scope of application of Halomonas cell factories. In summary, it is urgent to develop a quorum response mixed bacteria induction system based on interactive and stable expression of Halomonas, which can dynamically regulate target genes using cell density as a response signal, achieve induction in a mixed bacteria manner, and do not require exogenous addition of inducers, thereby reducing actual production costs and expanding the application prospects of Halomonas cell factories. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a method for constructing an interactive stable expression group response mixed bacteria induction system and its application in Halomonas.

[0005] The present invention discloses an interactive stable expression population response mixed bacteria induction system, wherein the interactive stable expression population response mixed bacteria induction system comprises a Plux-luxR-cinI induction expression system and a Pcin-cinR-luxI induction expression system;

[0006] The Plux-luxR-cinI inducible expression system comprises Plux, luxR, cinI and target gene;

[0007] The Pcin-cinR-luxI inducible expression system includes Pcin, cinR, luxI, and target gene;

[0008] The nucleotide sequence of luxR is shown in SEQ ID NO 1;

[0009] The nucleotide sequence of cinI is shown in SEQ ID NO 2;

[0010] The nucleotide sequence of cinR is shown in SEQ ID NO 3;

[0011] The nucleotide sequence of luxI is shown in SEQ ID NO 4;

[0012] The Plux is a promoter, which can be recognized and bound by the luxR transcriptional regulatory protein and the luxI signaling molecule protein complex, thereby regulating the expression of downstream target genes;

[0013] The luxR is a gene encoding a luxR transcriptional regulatory protein. In the Plux-luxR inducible expression system, the luxR transcriptional regulatory protein interacts with a signal molecule or an inducer (such as 3-oxohexanoyl-homoserine lactone, OC6) and binds to the Plux promoter;

[0014] The luxI is a gene encoding the signal molecule or inducer OC6 of the Plux-luxR inducible expression system;

[0015] The Pcin is a promoter, which can be recognized and bound by the cinR transcriptional regulatory protein and the cinI signal molecule protein complex, thereby regulating the expression of downstream target genes;

[0016] The cinR is a gene encoding a cinR transcriptional regulatory protein. In the Pcin-cinR inducible expression system, the cinR transcriptional regulatory protein interacts with a signal molecule or an inducer (such as 3-hydroxytetradecanoyl-homoserinelactone, OHC14) and binds to the Pcin promoter;

[0017] The cinI is a gene encoding the signal molecule or inducer OHC14 of the Pcin-cinR inducible expression system.

[0018] The target gene can be any gene selected according to the experimental purpose. In the embodiment of the present invention, the target gene is selected from Superfolder GFP (sfGFP) among fluorescent proteins.

[0019] Furthermore, the Plux is any one of a wild-type promoter Plux and a mutant promoter Plux;

[0020] The mutant promoter Plux is a wild-type promoter Plux-10 core region sequence TTGT mutated to any one of CATG, GGTG, CTGT, ATGT, and GTGG;

[0021] The nucleotide sequence of the wild-type promoter Plux is shown in SEQ ID NO 5.

[0022] Furthermore, the Pcin is any one of a wild-type promoter Pcin and a mutant promoter Pcin;

[0023] The mutant promoter Pcin is a wild-type promoter Pcin-10 core region sequence GAAC mutated to any one of TTAA, TCAT, GCCC, TGGG, and GGGG;

[0024] The nucleotide sequence of the wild-type promoter Pcin is shown in SEQ ID NO 6.

[0025] The present invention also provides a method for constructing the interactive stable expression population response mixed bacteria induction system, comprising the following steps:

[0026] (1) The construction method of the Plux-luxR-cinI inducible expression system is as follows:

[0027] (i) constructing Plux and luxR-cinI on the same plasmid to obtain a plasmid containing Plux-luxR-cinI;

[0028] or

[0029] Plux and luxR-cinI were constructed on different plasmids to obtain a plasmid containing Plux and a plasmid containing luxR-cinI;

[0030] (ii) transforming the plasmid containing Plux-luxR-cinI into Halomonas to obtain Halomonas containing the plasmid containing Plux-luxR-cinI;

[0031] or

[0032] The plasmid containing luxR-cinI is integrated into the genome of Halomonas to obtain Halomonas whose genome contains luxR-cinI, and then the plasmid containing Plux is transformed into Halomonas whose genome contains luxR-cinI to obtain Halomonas whose plasmid contains Plux and whose genome contains luxR-cinI; wherein luxR-cinI is integrated into the attp site of the Halomonas genome.

[0033] (2) The construction method of the Pcin-cinR-luxI inducible expression system is as follows:

[0034] (i) constructing Pcin and cinR-luxI on the same plasmid to obtain a plasmid containing Pcin-cinR-luxI;

[0035] or

[0036] Pcin and cinR-luxI were constructed on different plasmids to obtain a plasmid containing Pcin and a plasmid containing cinR-luxI;

[0037] (ii) transforming the plasmid containing Pcin-cinR-luxI into Halomonas to obtain Halomonas containing the plasmid containing Pcin-cinR-luxI;

[0038] or

[0039] The plasmid containing cinR-luxI is integrated into the genome of Halomonas to obtain Halomonas whose genome contains cinR-luxI, and then the plasmid containing Pcin is transformed into Halomonas whose genome contains cinR-luxI to obtain Halomonas whose plasmid contains Pcin and whose genome contains cinR-luxI; wherein luxR-cinI is integrated into the attp site of the Halomonas genome.

[0040] (3) Mixing and fermenting the Halomonas obtained in step (1) (ii) and the Halomonas obtained in step (2) (ii).

[0041] Furthermore, the Halomonas obtained in step (1) (ii) and the Halomonas obtained in step (2) (ii) are mixed in any volume ratio of 1:9, 1:4, 1:1, 4:1, and 9:1.

[0042] Preferably, the ratio of the mixed bacteria is (0.25-1):1, and the ratio of the mixed bacteria in the embodiment of the present invention is preferably 1:4.

[0043] Furthermore, the time for mixing the Halomonas obtained in step (1) (ii) and the Halomonas obtained in step (2) (ii) is 0h-16h.

[0044] The present invention also provides an interactive stable expression population response mixed bacteria induction expression amplification system, which comprises the Plux-luxR-cinI induction expression system, the Pcin-cinR-luxI induction expression system, a T7-like promoter in Halomonas, T7 RNA polymerase, and a target gene.

[0045] The present invention also provides a method for constructing the interactive stable expression population response mixed bacteria induction expression amplification system, comprising the following steps:

[0046] (1) replacing the pTac-RNAP promoter on the genome of the Halomonas bacteria containing cinR-luxI with the wild-type promoter Pcin to obtain the Halomonas bacteria containing the wild-type promoter Pcin in the genome;

[0047] (2) replacing the pTac-RNAP promoter on the genome of the Halomonas bacteria containing luxR-cinI with the wild-type promoter Plux, thereby obtaining Halomonas bacteria containing the wild-type promoter Plux in the genome;

[0048] (3) inserting the T7-like promoter and the nucleotide sequence of the target gene into the plasmid to obtain a recombinant plasmid;

[0049] Among them, the T7-like promoter selected in the embodiment of the present invention is the Pmmp1 promoter.

[0050] (4) The recombinant plasmid of step (3) is transformed into the Halomonas obtained in step (1) and the Halomonas obtained in step (2), respectively, and mixed bacteria and fermentation culture are performed.

[0051] Among them, the ratio of the mixed bacteria is preferably (0.25-1):1, and the ratio of the mixed bacteria in the embodiment of the present invention is preferably 1:4.

[0052] The present invention also provides an interactive stable expression population response mixed bacteria induced expression inhibition system, which comprises the Plux-luxR-cinI inducible expression system, the Pcin-cinR-luxI inducible expression system, dCas9, gRNA, and a target gene.

[0053] The present invention also provides a method for constructing the interactive stable expression population response mixed bacteria induced expression inhibition system, comprising the following steps:

[0054] (1) constructing a Pcin-dCas9-promoter-target gene expression module, and inserting the expression module into a plasmid to obtain a first plasmid;

[0055] Constructing a Pcin-gRNA expression module, and inserting the expression module into a plasmid to obtain a second plasmid;

[0056] The first plasmid and the second plasmid are co-transformed into the Halomonas whose genome contains cinR-luxI to obtain Halomonas with double plasmids;

[0057] (2) constructing a Plux-dCas9-promoter-target gene expression module, and inserting the expression module into the plasmid to obtain a third plasmid;

[0058] Constructing a Plux-gRNA expression module, inserting the expression module into the plasmid to obtain a fourth plasmid;

[0059] The third plasmid and the fourth plasmid are co-transformed into the Halomonas whose genome contains luxR-cinI to obtain Halomonas with double plasmids;

[0060] (3) Mixing and fermenting the Halomonas with double plasmids obtained in steps (1) and (2).

[0061] The present invention also provides the use of the interactive stable expression population response mixed bacteria induction system or the interactive stable expression population response mixed bacteria induction expression amplification system or the interactive stable expression population response mixed bacteria induction inhibition expression system in Halomonas.

[0062] In summary, compared with the prior art, the present invention has achieved the following technical effects: the interactive stable expression population response mixed bacteria induction system disclosed in the present invention, compared with the previous static regulation of metabolic pathways, can dynamically regulate the target gene with cell density as the response signal, and achieve induction by the mixed bacteria method, without the need for exogenous addition of inducers, thereby reducing the actual production cost and expanding the application prospects of the Halomonas cell factory. At the same time, based on the interactive stable expression population response mixed bacteria induction system, the amplified expression or inhibition of the target gene can be achieved, which can be used to study complex biological processes such as gene interactions, signal transduction pathways and biological regulatory networks. Through the interactive stable expression population response mixed bacteria induction system, it is possible to simulate cell-to-cell interactions and explore the regulatory mechanisms of different genes in complex environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0064] Figure 1 This is a plasmid information diagram of the reconnection of the Plux-sfGFP-Pporin-luxR and Pcin-sfGFP-Pporin-cinR fragments with the pSEVA321 plasmid backbone in Example 1 of the present invention;

[0065] Figure 2 This is a characterization result diagram of the Pcin-cinR induction system in Halomonas TD1.0 in Example 1 of the present invention;

[0066] Figure 3 This is a characterization result diagram of the Plux-luxR induction system in Halomonas TD1.0 in Example 1 of the present invention;

[0067] Figure 4 This is a plasmid information diagram of the pSEVA321-Pcin-sfGFP-Pporin-cinR and pSEVA321-Plux-sfGFP-Pporin-luxR plasmid backbones reconnected with the cinI and luxI fragments in Example 2 of the present invention;

[0068] Figure 5 This is a characterization result diagram of different mixed bacteria ratios of the Plux-luxR-cinI and Pcin-cinR-luxI induction expression systems in Example 2 of the present invention;

[0069] Figure 6 This is a characterization result diagram of different mixing times of the Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems in Example 2 of the present invention;

[0070] Figure 7 This is the plasmid information diagram used in Example 3 of the present invention;

[0071] Figure 8 This is a PCR verification result diagram of the attP sequence inserted into the Halomonas TD1.0 genome in Example 3 of the present invention;

[0072] Fig. 9 This is a PCR verification result diagram of the integration of Pporin-cinR-luxI and Pporin-luxR-cinI expression modules into the Halomonas TD1.0 genome in Example 3 of the present invention;

[0073] Fig.10 This is a characterization result diagram of the output signal intensity of the Pcin and Plux promoter mutation libraries in Example 4 of the present invention;

[0074] Fig.11 This is the information diagram of the PB 19-pSEVA321-Pcin-sfGFP and PB20-pSEVA321-Plux-sfGFP plasmids in Example 5 of the present invention;

[0075] Fig.12 This is a characterization result diagram of the interactive stable expression population response mixed bacteria induction system in Example 5 of the present invention;

[0076] Fig.13 This is a characterization result diagram of the interactive stable expression population response mixed bacteria induction system in a 7L fermenter in Example 6 of the present invention;

[0077] Fig.14 This is the plasmid information diagram used in Example 7 of the present invention;

[0078] Fig.15 This is a PCR verification result diagram of the promoter sequence replacement in Example 7 of the present invention;

[0079] Fig.16 This is a characterization result diagram of the target gene expression amplification system constructed based on the interactive stable expression population response mixed bacteria induction system in Example 7 of the present invention; wherein Fig.16 A is the average fluorescence intensity FI / OD of the mixed culture of the control group without amplification at 8h 600 picture; Fig.16 B is the average fluorescence intensity FI / OD of the mixed culture of the experimental group with amplified expression at 8h 600 picture; Fig.16C is the magnification comparison diagram at 8h;

[0080] Fig.17 Information diagram of plasmid used in Example 8 of the present invention;

[0081] Fig.18 A diagram showing the characterization results of the target gene expression inhibition system constructed based on the interactive stable expression population response mixed bacteria induction system in Example 8 of the present invention. DETAILED DESCRIPTION

[0082] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0083] Example 1 Construction and characterization of Plux-luxR and Pcin-cinR inducible expression systems

[0084] 1. Construction of Plux-luxR and Pcin-cinR inducible expression system plasmids

[0085] Plux-sfGFP-Pporin-luxR and Pcin-sfGFP-Pporin-cinR fragments were obtained by gene synthesis, and PCR amplification was performed using the synthetic plasmid as a template to obtain linearized Plux-sfGFP-Pporin-luxR and Pcin-sfGFP-Pporin-cinR fragments; PCR amplification was performed using pSEVA321-Pporin58-sfGFP as a template to obtain the linearized pSEVA321 plasmid backbone fragment, and the fragments were amplified by Gibson. Assembly kit, and the linearized Plux-sfGFP-Pporin-luxR and Pcin-sfGFP-Pporin-cinR fragments were connected with the linearized pSEVA321 plasmid backbone fragment, respectively, to obtain two new plasmids named PY47-pSEVA321-Pcin-sfGFP-Pporin-cinR plasmid (referred to as PY47) and PY75-pSEVA321-Plux-sfGFP-Pporin-luxR plasmid (referred to as PY75). The constructed PY47 and PY75 plasmids were used as templates for PCR verification, and Sanger sequencing was performed to verify the correctness of the sequence. The plasmid information is as follows: Figure 1 shown.

[0086] The primer sequences (5'-3') for amplifying the linearized Plux-sfGFP-Pporin-luxR, Pcin-sfGFP-Pporin-cinR fragments and the pSEVA321 plasmid backbone are as follows:

[0087] 47-Frag.F: see SEQ ID No.7; 47-Frag.R: see SEQ ID No.8;

[0088] 47-Vec.F: see SEQ ID No.9; 47-Vec.R: see SEQ ID No.10;

[0089] 75-Frag.F: see SEQ ID No.11; 75-Frag.R: see SEQ ID No.12;

[0090] 75-Vec.F: see SEQ ID No.13; 75-Vec.R: see SEQ ID No.14;

[0091] The amplification system and amplification procedure are shown in Table 1 and Table 2:

[0092] Table 1 PCR amplification system

[0093]

[0094] Table 2 PCR amplification program

[0095]

[0096] After the PCR reaction, 1% agarose gel was prepared for electrophoresis separation of PCR products. The agarose gel after electrophoresis was placed in a blue light gel exciter, and the gel block with the target DNA fragment located in the correct position was cut out. The target DNA fragment was recovered using a universal DNA purification and recovery kit (Tian Gen). The concentration of the recovered DNA product was detected using Nano-400A. The addition ratio of each DNA fragment was calculated according to the Gibson Assembly kit instructions, and Gibson Assembly connection was performed. The Gibson Assembly connection system and connection procedure are shown in Tables 3 and 4:

[0097] Table 3 Gibson Assembly connection system

[0098]

[0099] Table 4 Gibson Assembly connection procedure

[0100]

[0101] After the ligation procedure is completed, 10 μL of the ligation product is added to 50 μL of E. coli S17-1 competent cells, placed in an ice bath for 30 minutes, heat-shocked in a 42°C water bath for 1 minute and 30 seconds, and then immediately placed on ice for 2 minutes. 450 μL of LB medium (without antibiotics) is added to the centrifuge tube, mixed and placed in a 37°C 200rpm shaker for 60 minutes. Centrifuge and concentrate, spread on an LB plate containing the corresponding resistance, and place in a 37°C incubator overnight. After a monoclonal colony grows, perform colony PCR verification, and perform Sanger sequencing on the PCR product with the correct band size to verify the correctness of the plasmid sequence.

[0102] 2. Characterization of different inducer concentration gradients

[0103] (1) Plasmid conjugation transformation of Halomonas TD1.0

[0104] E.coli S17-1 containing PY47 and PY75 plasmids were expanded overnight in LB medium with corresponding resistance, and Halomonas TD1.0 was expanded in 60LB medium without antibiotics, then combined on 20LB plates and cultured in a 37°C incubator for 8-12 hours. A small amount of bacteria was scraped and spread on the 60LB plate with corresponding resistance. After about 36-48 hours, monoclonal colonies of Halomonas TD1.0 containing plasmids could be grown.

[0105] (2) Deep-well plate culture of Halomonas TD1.0

[0106] The Halomonas TD1.0 monoclonal colony with the plasmid in (1) was streaked and activated on a new 60LB plate with the same resistance. A small amount of activated bacteria was picked and cultured in a deep-well plate, inoculated into a well containing 1 mL of 60LB culture medium with the corresponding resistance as the primary seed liquid, and cultured for 12 hours; the primary seed liquid was transferred to a new well containing 1 mL of 60LB culture medium with the corresponding resistance as the secondary seed liquid, and cultured for 12 hours. The secondary seed liquid was transferred to a new well containing 1 mL of 60LB culture medium with the corresponding resistance for formal culture. Each experimental group was set up with three wells of biological replicate parallel experiments to ensure the reliability of the data. The deep-well plate was cultured at 37°C and 1000rpm using a Thermo Shaker (MB100-4A).

[0107] (3) Validation of the Plux-luxR and Pcin-cinR induction systems in Halomonas TD1.0

[0108] The Plux-luxR and Pcin-cinR induction systems of Halomonas TD1.0 were experimentally verified in deep-well plates using the deep-well plate culture method mentioned in (2). -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 The final concentration gradient of the inducer was 100 mM, and three wells were set for each concentration gradient for biological replicate parallel experiments. The inducer of the Pcin-cinR induction system was OHC14 (3-hydroxytetradecanoyl-homoserine lactone), and the inducer of the Plux-luxR induction system was OC6 (3-oxohexanoyl-homoserine lactone). The inducer was added at 0h of formal culture, and samples were taken and diluted at 12h and 24h of formal culture. The fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at a wavelength of 520nm when the excitation wavelength is 488nm) and OD of the culture were detected using a Thermo Varioskan Lux microplate reader. 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 as a characterization indicator.

[0109] (4) Characterization results of the Plux-luxR and Pcin-cinR induction systems in Halomonas TD1.0

[0110] The experiment was carried out using the verification scheme in (3), and the results are as follows Figure 2 , Figure 3 As shown in Figure 2, the Plux-luxR and Pcin-cinR induction systems in Halomonas TD1.0 have been successfully constructed and can induce the expression of sfGFP by adding exogenous inducers. The characterization results of the two induction systems are shown in Figure 2. Figure 2 , Figure 3 The results showed that the minimum response concentration of the Pcin-cinR induction system to the OHC14 inducer was 10 -6 ~10 -5 mM, at 10 -2 The induction effect was best at a concentration of 10 mM. The lowest response concentration of the Plux-luxR induction system to OC6 inducer was 10 -5 ~10 -4 mM, at 10 -2 The induction effect was best at mM concentration.

[0111] Example 2 Construction and characterization of Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems

[0112] 1. Construction of Plux-luxR-cinI and Pcin-cinR-luxI expression plasmids

[0113] The specific implementation of plasmid construction is the same as that of Example 1-1. The linearized pSEVA321-Pcin-sfGFP-Pporin-cinR plasmid backbone fragment is amplified using the PY47 plasmid as a template, and the linearized pSEVA321-Plux-sfGFP-Pporin-luxR plasmid backbone fragment is amplified using the PY75 plasmid as a template. The cinI and luxI fragments are obtained by gene synthesis. The cinI and luxI fragments are amplified using the synthetic plasmid as a template to obtain two new plasmids, respectively named PY29-pSEVA321-Pcin-sfGFP-Pporin-cinR-luxI plasmid (abbreviated as PY29) and PY34-pSEVA321-Plux-sfGFP-Pporin-luxR-cinI plasmid (abbreviated as PY34). The plasmid information is as follows: Figure 4 shown.

[0114] The amplification primer sequences (5'-3') are as follows:

[0115] 29-Frag.F: see SEQ ID No.15; 29-Frag.R: see SEQ ID No.16;

[0116] 29-Vec.F: see SEQ ID No.17; 29-Vec.R: see SEQ ID No.18;

[0117] 34-Frag.F: see SEQ ID No.19; 34-Frag.R: see SEQ ID No.20;

[0118] 34-Vec.F: see SEQ ID No.21; 34-Vec.R: see SEQ ID No.22;

[0119] 2. Characterization of different mixed bacterial ratios

[0120] The cinI gene in Example 2-1 can produce OHC14 inducer, and the OHC14 inducer is the inducer of the Pcin-cinR induction system; the luxI gene can produce OC6 inducer, and the OC6 inducer is the inducer of the Plux-luxR induction system; the concentration of the inducer produced by the cinI gene and the luxI gene can be considered to be positively correlated with the cell density, and the produced inducer has membrane permeability. Based on the above characteristics, the construction of an interactive and stable expression group response mixed bacteria induction system can be achieved by mixing bacteria instead of adding exogenous inducers.

[0121] Plasmid conjugation transformation, deep-well plate culture, and characterization of the Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems in Halomonas TD1.0 were performed as described in Example 1. The characterization methods for different mixed bacterial ratios are as follows:

[0122] At 0h of formal culture, 900μL of Halomonas TD1.0 culture with PY29 plasmid (referred to as culture A) and 100μL of Halomonas TD1.0 culture with PY34 plasmid (referred to as culture B) were taken out, and 900μL of culture A and 100μL of culture B were mixed and induced in a new well. Another 900μL of culture B and 100μL of culture A were taken out and mixed and induced in a new well. This is a 1:9 mixed ratio group. In the same way as above, 1:4 and 1:1 mixed ratio groups were set up. Each mixed ratio experimental group was set up with three wells of biological replicate parallel experiments. Samples were taken and diluted at 12h and 24h of formal culture, and the fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at a wavelength of 520nm when the excitation wavelength is 488nm) and OD of the culture were detected using a Thermo Varioskan Lux microplate reader. 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 As a characterization index. The characterization results of different mixed bacteria ratios of Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems are shown in Figure 5 As shown, the Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems can achieve the induced expression of sfGFP in Halomonas TD1.0 by mixing bacteria, and the induction effect of the 1:4 mixed bacteria ratio group is better.

[0123] 3. Characterization of different mixing times

[0124] Plasmid conjugation transformation, deep-well plate culture, and characterization of the Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems in Halomonas TD1.0 were performed as described in Example 1. The characterization methods for different mixing times are as follows:

[0125] At 0h, 8h, and 12h of formal culture, 800μL of Halomonas TD1.0 culture with PY29 plasmid (referred to as culture A) and 200μL of Halomonas TD1.0 culture with PY34 plasmid (referred to as culture B) were taken out, and 800μL of culture A and 200μL of culture B were mixed and induced in new wells, and 800μL of culture B and 200μL of culture A were taken out and mixed and induced in new wells. Three wells of biological replicate parallel experiments were set up for each mixed time experimental group. Samples were taken and diluted at 12h, 24h, and 36h of formal culture, and the fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at a wavelength of 520nm when the excitation wavelength is 488nm) and OD of the culture were detected using a Thermo Varioskan Lux microplate reader. 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 The characterization results of different mixing times of Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems are shown in the figure. Figure 6 As shown: Plux-luxR-cinI and Pcin-cinR-luxI inducible expression systems were mixed in Halomonas TD1.0 at a ratio of 1:4 at 0h, 8h, and 12h of formal culture, and both were able to induce the expression of sfGFP. The induction effect was best when mixed at 0h. In practical applications, the mixing induction time can be flexibly selected according to specific circumstances.

[0126] Example 3 Construction and verification of luxR-cinI and cinR-luxI genomic expression plasmids

[0127] 1. Construction of integration plasmid

[0128] In order to improve the stability of the interactively stably expressed quorum-responsive mixed-bacteria induction system in Halomonas TD1.0 and simplify its gene circuit design in practical applications, the Pporin-luxR-cinI and Pporin-cinR-luxI expression modules were integrated into the Halomonas TD1.0 genome, and the integrated strains were named TY01 (Halomonas TD1.0 with Pporin-cinR-luxI expression module) and TY02 (Halomonas TD1.0 with Pporin-luxR-cinI expression module). In this example, the expression module was integrated into the genome by combining the CRISPR-Cas9 method with the integrase gene integration method. The specific implementation of the plasmid construction was the same as that of Example 1-1. The G53Ldonor and G53Rdonor fragments were amplified using the Halomonas TD1.0 genome as a template. According to the method of Example 1, the G53Ldonor and G53Rdonor fragments were ligated into the pSEVA341 vector to successfully construct pSEVA341-G53Ldonor-attP-G53Rdonor. The constructed plasmid was amplified, and the PCR products were ligated using T4 ligase to recover the fragments. The T4 ligase reaction system and procedure are detailed in Tables 5 and 6. pSEVA341-G53gRNA-G53Ldonor-attP-G53Rdonor was successfully constructed, and pSEVA321-Cas9 and pSEVA341-G53gRNA-G53Ldonor-attP-G53Rdonor vectors were introduced into Halomonas TD1.0. attP sequence was inserted into the TD1.0 genome; the expression module integration fragment was obtained by gene synthesis, and the synthetic plasmid was used as a template to amplify the SloxP-attB-Pporin-cinR-luxI-SloxP-pTac-Int5 and SloxP-attB-Pporin-luxR-cinI-SloxP-pTac-Int5 fragments. According to the method of Example 1, the SloxP-attB-Pporin-cinR-luxI-SloxP-pTac-Int5 and SloxP-attB-Pporin-luxR-cinI-SloxP-pTac-Int5 fragments were connected to the pSEVA321 vector. The two newly constructed plasmids were named YP45 and YP46, and the two expression modules Pporin-cinR-luxI and Pporin-luxR-cinI were integrated into the Halomonas TD1.0 genome by the integrase gene integration method. The above plasmid information is as follows Figure 7 shown.

[0129] The T4 ligase reaction system and procedure are shown in Table 5 and Table 6:

[0130] Table 5 T4 ligase ligation system

[0131]

[0132]

[0133] Table 6 T4 ligase ligation procedure

[0134]

[0135] The amplification primer sequences (5'-3') used in Example 3 are as follows:

[0136] G53L-F: see SEQ ID No. 23; G53L-R: see SEQ ID No. 24;

[0137] G53R-F: see SEQ ID No. 25; G53R-R: see SEQ ID No. 26;

[0138] 341-Vec.F: see SEQ ID No.27; 341-Vec.R: see SEQ ID No.28;

[0139] G53-sgRNA.F: see SEQ ID No.29; G53-sgRNA.R: see SEQ ID No.30;

[0140] 45 / 46-Frag.F: see SEQ ID No.31; 45 / 46-Frag.R: see SEQ ID No.32;

[0141] 45 / 46-Vec.F: see SEQ ID No.33; 45 / 46-Vec.R: see SEQ ID No.34;

[0142] 2. Genome Integration and Verification

[0143] (1) Insertion and verification of attP sites in the Halomonas TD1.0 genome

[0144] The attP sequence in the Halomonas TD1.0 genome was inserted using the CRISPR / Cas9 method and verified by designing primers on the attP sequence and upstream of G53Ldonor, such as Figure 8 The agarose electrophoresis shown shows a target band of 1100 bp, indicating that the attP sequence was successfully inserted into the Halomonas TD1.0 genome.

[0145] (2) Integration and verification of Pporin-cinR-luxI and Pporin-luxR-cinI expression modules on the Halomonas TD1.0 genome

[0146] The two expression modules Pporin-cinR-luxI and Pporin-luxR-cinI were integrated into the Halomonas TD1.0 genome using the integrase gene integration method, and the primers were designed within the inserted gene and upstream of G53Ldonor for verification. Fig. 9 As shown, agarose electrophoresis showed a 2000 bp band, indicating that the expression module was successfully integrated into the Halomonas TD1.0 genome. Halomonas TD1.0 integrated with the Pporin-cinR-luxI expression module was named TY01, and Halomonas TD1.0 integrated with the Pporin-luxR-cinI expression module was named TY02.

[0147] Example 4 Construction and characterization of Plux and Pcin promoter libraries

[0148] 1. Random mutagenesis of the promoter core region

[0149] In order to expand the signal output range of the interactive stable expression group response mixed bacteria induction system, it is necessary to obtain Pcin and Plux promoter libraries of different strengths. By randomly mutating the first 4 bases of the Pcin and Plux promoter-10 core region sequence, Pcin_NNNN and Plux_NNNN promoter libraries of different output strengths can be obtained (N refers to any base of A, T, C, G). The plasmid construction method is referenced to Example 1. The first 4 bases of the promoter-10 core region sequence are randomly mutated by introducing random mutations with primers. PCR is performed using PY47 and PY75 plasmids as templates, and T4 ligase is used to connect the PCR product recovery fragments to screen out a series of Pcin and Plux promoter mutation libraries (PY47-Pcin_NNNN and PY75-Plux_NNNN plasmid libraries, respectively) that respond to exogenous addition of inducers.

[0150] The random mutation primer sequences (5'-3') are as follows:

[0151] Pcin-random.F: see SEQ ID No.35;

[0152] Pcin-R: see SEQ ID No.36;

[0153] Plux-random.F: see SEQ ID No.37;

[0154] Plux-R: see SEQ ID No.38;

[0155] The first 4 base sequences (5'-3') of the core region of the obtained Pcin and Plux promoter mutation library-10 are shown in Table 7:

[0156] Table 7 Pcin and Plux promoter mutation library-10 core region first 4 base sequences

[0157]

[0158]

[0159] 2. Mutation Characterization

[0160] After obtaining the Pcin and Plux promoter mutant libraries, the output signal strength was characterized by exogenously adding inducers. The plasmid binding transformation, deep-well plate culture, and characterization results in Halomonas TD1.0 were referred to Example 1. The output signal strength of the Pcin and Plux promoter mutant libraries in Halomonas TD1.0 was characterized in a deep-well plate. During formal culture, 10 -2 The final concentration gradient of the inducer was 100 mM, and three wells were set up for each mutation. The inducer was added at 0h of formal culture, and samples were taken and diluted at 12h of formal culture. The fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at 520nm when the excitation wavelength is 488nm) and OD 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 The characterization results of the output signal intensity of the Pcin and Plux promoter mutation libraries are as follows: Fig.10 As shown, the output signal intensity of Pcin_GGGG, Pcin_TGGG, and Pcin_GCCC after mutation is stronger than that of Pcin_GAAC(wt), and the output signal intensity of Pcin_TCAT and Pcin_TTAA after mutation is weaker than that of Pcin_GAAC(wt); the output signal intensity of Plux_GTGG, Plux_ATGT, and Plux_CTGT after mutation is stronger than that of Plux_TTGT(wt), and the output signal intensity of Plux_GGTG and Plux_CATG after mutation is weaker than that of Plux_TTGT(wt). In practical applications, Pcin / Plux promoter mutations with different output signal intensities can be selected according to specific circumstances.

[0161] Example 5 Testing of the interactive mixed bacteria induction system in Halomonas TD1.0

[0162] 1. Construction of test plasmid for interactive mixed bacteria induction system

[0163] After obtaining TY01 and TY02 strains from Example 3, since the two expression modules Pporin-cinR-luxI and Pporin-luxR-cinI are expressed on the genome, the plasmid only needs to have the target gene expression module controlled by Pcin or Plux to achieve mixed bacterial induction. The target gene in this example is sfGFP. The specific implementation of the plasmid construction is as shown in Example 1-1. PY47 and PY75 are used as templates for amplification, and T4 ligase is used to connect the PCR product to recover the fragments to obtain the newly constructed PB19pSEVA321-Pcin-sfGFP and PB20-pSEVA321-Plux-sfGFP plasmids, which are named PB19 and PB20, respectively. The plasmid information is as follows: Fig.11 shown.

[0164] The plasmid amplification primer sequences (5'-3') are as follows:

[0165] PB19-F: see SEQ ID No.39; PB19-R: see SEQ ID No.40;

[0166] PB20-F: see SEQ ID No.41; PB20-R: see SEQ ID No.42;

[0167] 2. Characterization of the interactive mixed-bacteria induction system

[0168] The plasmid binding transformation, deep-well plate culture, and characterization results of the interactive mixed bacteria induction expression system in TY01 and TY02 were referred to Example 1. The characterization method in this example is as follows:

[0169] At 0h, 8h, and 12h of formal culture, 800μL of TY01 culture with PB19 plasmid (referred to as culture C) and 200μL of TY02 culture with PB20 plasmid (referred to as culture D) were taken out, and 800μL of culture C and 200μL of culture D were mixed and induced in a new well, and 800μL of culture B and 200μL of culture A were taken out and mixed and induced in a new well, that is, they were mixed in a volume ratio of 1:4, and three wells of biological replicate parallel experiments were set up for each mixed time experimental group. Samples were taken and diluted at 12h, 24h, and 36h of formal culture, and the fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at a wavelength of 520nm when the excitation wavelength is 488nm) and OD of the culture were detected using a Thermo Varioskan Lux microplate reader. 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 As a characterization index. The characterization results of the interactive mixed bacteria induction expression system are as follows Fig.12 As shown, the interactive mixed bacteria induction expression system can induce the expression of sfGFP in TY01 and TY02 at a ratio of 1:4 when the bacteria are mixed at 0h, 8h, and 12h of formal culture.

[0170] Example 6 Testing of the interactive stable expression population response mixed bacteria induction system in a fermenter

[0171] Culture system and protocol in 1.7L fermenter

[0172] The strain-plasmid combination (TY01-PB19, TY02-PB20) in Example 5 was used to test the interactive stable expression population response mixed bacteria induction system in a 7L fermenter. The plasmid binding transformation and characterization results in TY01 and TY02 were referred to Example 1. The fermentation scheme and culture system in this example are as follows:

[0173] (1) Fermentation plan for 7L fermenter

[0174] TY01-PB19 and TY02-PB20 monoclonal colonies were streaked and activated on a new 60LB plate with the same resistance. A small amount of activated bacteria was picked and inoculated into a 12mL shaking tube containing 4mL of 60LB medium with corresponding resistance as the first-level seed liquid, and cultured for 12 hours; the second-level seed liquid was transferred to a new 150mL shaking flask containing 20mL of 60LB medium with corresponding resistance as the second-level seed liquid, and cultured for 12 hours. The third-level seed liquid was transferred to a new 500mL shaking flask containing 100mL of 60LB medium with corresponding resistance as the third-level seed liquid, and cultured for 12 hours. The third-level seed liquid was added to a 7L fermentation tank containing 4L fermentation substrate at a ratio of 10% for formal culture, of which TY01-PB19 was tank No. 1 and TY02-PB20 was tank No. 2. After 8 hours of formal culture, 800 mL of TY01 culture with PB19 plasmid (referred to as culture C) was taken out from tank No. 1, and 800 mL of TY02 culture with PB20 plasmid (referred to as culture D) was taken out from tank No. 2. 800 mL of culture C was poured into tank No. 2, and 800 mL of culture D was poured into tank No. 1 for mixed bacteria induction culture. Samples were taken and diluted every hour, and the fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at a wavelength of 520 nm when the excitation wavelength is 488 nm) and OD of the culture were detected using a Thermo Varioskan Lux microplate reader. 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 as a characterization indicator.

[0175] (2) The 7L fermentation tank culture system adopts the formula in the following literature:

[0176] Pilot Scale-up of Poly(3-hydroxybutyrate-co-4hydroxybutyrate)Production by Halomonas bluephagenesis via Cell Growth Adapted OptimizationProcess:

[0177] 2. Characterization results of the interactive stable expression population response mixed bacteria induction system in a fermenter

[0178] The test results of the interactive stable expression population response mixed bacteria induction system in a 7L fermenter are as follows Fig.13 As shown, when a 7L fermenter system is used for fermentation culture, the interactive mixed bacteria induction expression system can induce the expression of sfGFP by mixing TY01 and TY02 at a ratio of 1:4 after 8 hours of formal culture.

[0179] Example 7 Inducible expression of T7 RNA polymerase to achieve amplified expression of GFP

[0180] 1. Construction of relevant plasmids for target gene expression amplification system

[0181] The purpose of this embodiment is to achieve expression amplification of the target gene by controlling the expression of T7 RNA polymerase with Pcin and Plux promoters and controlling the expression of the target gene with Pmmp1 promoter (T7-like promoter in Halomonas) through the T7-like expression system in Halomonas.

[0182] In this embodiment, the promoter of the pTac-RNAP expression module on the TY01 and TY02 genomes was replaced with Pcin and Plux respectively using the CRISPR / Cas9 method. The pTac-donorL and pTac-donorR fragments were amplified using the Halomonas TD1.0 genome as a template, the Pcin fragment was amplified using PB19 as a template, and the Plux fragment was amplified using PB20 as a template. The specific operation was referred to Example 1, and the pTac-donorL and pTac-donorR fragments were connected to the pSEVA341 vector with the Pcin and Plux fragments, respectively, and pSEVA341-pTac-donorL-Pcin-pTac-donorR and pSEVA341-pTac-donorL-Plux-pTac-donorR were successfully constructed. The constructed plasmids were amplified, and the PCR products were connected using T4 ligase to recover the fragments. pSEVA341-pTac-gRNA-pTac-donorL-Pcin-pTac-donorR and pSEVA341-pTac-gRNA-pTac-donorL-Plux-pTac-donorR were successfully constructed. The pSEVA321-Cas9 and pSEVA341-pTac-gRNA-pTac-donorL-Pcin-pTac-donorR vectors were introduced into TY01, and the promoter of the pTac-RNAP expression module on the TY01 genome was successfully replaced with Pcin, named TY01-Pcin; the pSEVA321-Cas9 and pSEVA341-pTac-gRNA-pTac-donorL-Plux-pTac-donorR vectors were introduced into TY02, and the promoter of the pTac-RNAP expression module on the TY02 genome was successfully replaced with Plux, named TY02-Plux. Referring to the method of Example 1, pSEVA321-Pporin58-sfGFP plasmid was used as a template for amplification, and T4 ligase was used to connect the PCR product to recover the fragments, and pSEVA321-Pmmp1-sfGFP plasmid was successfully constructed. The plasmid information described in this example is as follows Fig.14 shown.

[0183] The plasmid construction primer sequences (5'-3') are as follows:

[0184] pTac-donorL-F: see SEQ ID No.43; pTac-donorL-R: see SEQ ID No.44;

[0185] pTac-donorR-F: see SEQ ID No.45; pTac-donorR-R: see SEQ ID No.46;

[0186] 341-Vec.F: see SEQ ID No.47; 341-Vec.R: see SEQ ID No.48;

[0187] Pcin-F: see SEQ ID No.49; Pcin-R: see SEQ ID No.50;

[0188] Plux-F: see SEQ ID No.51; Plux-R: see SEQ ID No.52;

[0189] pTac-sgRNA-F: see SEQ ID No.53; pTac-sgRNA-R: see SEQ ID No.54;

[0190] Pmmp1-F: see SEQ ID No.55; Pmmp1-R: see SEQ ID No.56;

[0191] The promoter sequence replacement was verified by PCR by designing primers on the inserted genomic sequence and downstream of pTac-donorR. The results were as follows Fig.15 As shown, agarose electrophoresis showed a 1300 bp band, indicating that the Pcin promoter sequence was successfully replaced; agarose electrophoresis showed a 1500 bp band, indicating that the Plux promoter sequence was successfully replaced.

[0192] 2. Test and characterization results of the target gene expression amplification system

[0193] The expression of the target gene was amplified in TY01, TY02, TY01-Pcin, and TY02-Plux. The plasmid binding transformation, deep-well plate culture, and characterization results were described in Example 1. The characterization method in this example is as follows:

[0194] The PB19 plasmid was introduced into TY01 and the PB20 plasmid was introduced into TY02 as the control group without amplification, and the pSEVA321-Pmmp1-sfGFP plasmid was introduced into TY01-Pcin and TY02-Plux respectively as the experimental groups with expression amplification.

[0195] At 0h, 8h, and 12h of formal culture, 800μL of TY01 culture with PB19 plasmid (hereinafter referred to as culture C) and 200μL of TY02 culture with PB20 plasmid (hereinafter referred to as culture D) were taken out, and 800μL of culture C and 200μL of culture D were mixed in a new well for induction culture, and another 800μL of culture D and 200μL of culture C were taken out and mixed in a new well for induction culture; at 0h, 8h, and 12h of formal culture, 800μL of culture D with pSEVA32 plasmid were taken out and cultured. 1-Pmmp1-sfGFP plasmid TY01-Pcin culture (hereinafter referred to as culture E) and 200 μL of TY02-Plux culture (hereinafter referred to as culture F) with pSEVA321-Pmmp1-sfGFP plasmid, 800 μL of culture E and 200 μL of culture F were mixed and induced in new wells, and another 800 μL of culture F and 200 μL of culture E were taken out and mixed and induced in new wells. For each mixing time experimental group, three wells were set up for biological replicate parallel experiments. At 12h of secondary seed culture, 8h and 12h of formal culture, samples were taken and diluted as controls. Samples were taken and diluted 12 and 24h after each mixing time point (0h / 8h / 12h+12h / 24h). The fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at a wavelength of 520nm when the excitation wavelength is 488nm) and OD of the culture were detected using a Thermo Varioskan Lux microplate reader. 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 As a characterization index. The characterization results of the target gene expression amplification system are as follows Fig.16 As shown: The target gene expression amplification system constructed based on the interactive mixed bacteria induced expression system can effectively amplify the expression of the target gene (sfGFP), and the amplification effect is good, and can achieve more than 20 times of expression amplification.

[0196] Example 8: Inhibition of target gene expression by suppressing system

[0197] 1. Construction of relevant plasmids of target gene expression inhibition system

[0198] The purpose of this example is to construct a target gene expression inhibition system based on the interactive mixed bacteria induction expression system through the CRISPRi method, using Pcin and Plux to control the expression of dCas9 and the transcription of gRNA, respectively, and using Pporin58 to control the expression of the target gene (sfGFP), and to achieve the expression inhibition of the target gene by inducing the expression of dCas9 and the transcription of gRNA through mixed bacteria.

[0199] Pcin-dCas9 and Plux-dCas9 fragments were obtained by gene synthesis, and PCR amplification was performed using the synthetic plasmid as a template to obtain Pcin-dCas9 and Plux-dCas9 fragments, and PCR amplification was performed using pSEVA321-Pporin58-sfGFP as a template to obtain the pSEVA321-Pporin58-sfGFP skeleton, which was connected with the Pcin-dCas9 and Plux-dCas9 fragments using the Gbison Assembly kit to construct pSEVA321-Pcin-dCas9-Pporin58-sfGFP and pSEVA321-Plux-dCas9-Pporin58-sfGFP plasmids. Pcin-gRNA and Plux-gRNA fragments were obtained by gene synthesis, and PCR amplification was performed using the synthetic plasmid as a template to obtain Pcin-gRNA and Plux-gRNA fragments, which were then connected to the pSEVA341 skeleton to construct pSEVA341-Pcin-gRNA and pSEVA341-Plux-gRNA plasmids. The plasmid information described in this example is as follows Fig.17 shown.

[0200] The amplification primer sequences (5'-3') used for the plasmid described in this example are as follows:

[0201] Pcin-dCas9-F: see SEQ ID No.57; Plux-dCas9-F: see SEQ ID No.58;

[0202] dCas9-R: see SEQ ID No.59; dCas9-Vec.F: see SEQ ID No.60;

[0203] dCas9-Vec.R: see SEQ ID No.61; gRNA-F: see SEQ ID No.62;

[0204] gRNA-R: see SEQ ID No.63; gRNA-Vec.F: see SEQ ID No.64;

[0205] gRNA-Vec.R: see SEQ ID No.65;

[0206] 2. Test and characterization results of the target gene expression inhibition system

[0207] The expression inhibition of the target gene in TY01 and TY02 was carried out by plasmid binding transformation, deep well plate culture, and characterization of the results. The method is as follows:

[0208] The pSEVA321-Pcin-dCas9-Pporin58-sfGFP plasmid and pSEVA341-Pcin-gRNA plasmid were introduced into TY01, and the pSEVA321-Plux-dCas9-Pporin58-sfGFP and pSEVA341-Plux-gRNA plasmids were introduced into TY02.

[0209] At 0h, 8h, and 12h of formal culture, 800μL of TY01 culture with double plasmids (hereinafter referred to as culture G) and 200μL of TY02 culture with double plasmids (hereinafter referred to as culture H) were taken out, and 800μL of culture G and 200μL of culture H were mixed and induced in new wells, and 800μL of culture H and 200μL of culture G were taken out and mixed and induced in new wells. Samples were taken and diluted after mixing at 12h of secondary seed liquid culture and 8h and 12h of formal culture as controls. Samples were taken and diluted 12h and 24h after each mixing time point (0h / 8h / 12h+12h / 24h), and the fluorescence intensity FI 488 / 520 (referring to the fluorescence emission intensity at a wavelength of 520nm when the excitation wavelength is 488nm) and OD of the culture were detected using a Thermo Varioskan Lux microplate reader. 600 (The absorbance at a wavelength of 600 nm reflects the bacterial growth state and bacterial density), and the average fluorescence intensity FI / OD of the culture is calculated. 600 As a characterization index. The characterization results of the target gene expression inhibition system are as follows Fig.18 As shown: The target gene expression inhibition system constructed based on the interactive mixed bacteria induction expression system can effectively inhibit the expression of the target gene (sfGFP), and the average fluorescence intensity of the culture can be reduced by 2-6 times during 24h cultivation.

[0210] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An interactive stable expression population response mixed bacteria induction system, characterized in that: The interactive stable expression population response mixed bacteria induction system includes a Plux-luxR-cinI induction expression system and a Pcin-cinR-luxI induction expression system; The Plux-luxR-cinI inducible expression system comprises Plux, luxR, cinI and a target gene; The Pcin-cinR-luxI inducible expression system comprises Pcin, cinR, luxI and target gene; The nucleotide sequence of luxR is shown in SEQ ID NO 1; The nucleotide sequence of cinI is shown in SEQ ID NO 2; The nucleotide sequence of cinR is shown in SEQ ID NO 3; The nucleotide sequence of luxI is shown as SEQ ID NO 4.

2. The interactive stable expression population response mixed bacteria induction system according to claim 1, characterized in that: The Plux is any one of a wild-type promoter Plux and a mutant promoter Plux; The mutant promoter Plux is a wild-type promoter Plux-10 core region sequence TTGT mutated to any one of CATG, GGTG, CTGT, ATGT, and GTGG; The nucleotide sequence of the wild-type promoter Plux is shown in SEQ ID NO 5.

3. The interactive stable expression population response mixed bacteria induction system according to claim 1, characterized in that: The Pcin is any one of a wild-type promoter Pcin and a mutant promoter Pcin; The mutant promoter Pcin is a wild-type promoter Pcin-10 core region sequence GAAC mutated to any one of TTAA, TCAT, GCCC, TGGG, and GGGG; The nucleotide sequence of the wild-type promoter Pcin is shown in SEQ ID NO 6.

4. The method for constructing an interactive stable expression population response mixed bacteria induction system according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) The construction method of the Plux-luxR-cinI inducible expression system is as follows: (i) constructing Plux and luxR-cinI on the same plasmid to obtain a plasmid containing Plux-luxR-cinI; or Plux and luxR-cinI were constructed on different plasmids to obtain a plasmid containing Plux and a plasmid containing luxR-cinI; (ii) transforming the plasmid containing Plux-luxR-cinI into Halomonas to obtain Halomonas containing the plasmid containing Plux-luxR-cinI; or The plasmid containing luxR-cinI is integrated into the genome of Halomonas to obtain Halomonas with luxR-cinI in the genome, and then the plasmid containing Plux is transformed into Halomonas with luxR-cinI in the genome to obtain Halomonas with the plasmid containing Plux and luxR-cinI in the genome; (2) The construction method of the Pcin-cinR-luxI inducible expression system is as follows: (i) constructing Pcin and cinR-luxI on the same plasmid to obtain a plasmid containing Pcin-cinR-luxI; or Pcin and cinR-luxI were constructed on different plasmids to obtain a plasmid containing Pcin and a plasmid containing cinR-luxI; (ii) transforming the plasmid containing Pcin-cinR-luxI into Halomonas to obtain Halomonas containing the plasmid containing Pcin-cinR-luxI; or The plasmid containing cinR-luxI is integrated into the genome of Halomonas to obtain Halomonas with cinR-luxI in the genome, and then the plasmid containing Pcin is transformed into Halomonas with cinR-luxI in the genome to obtain Halomonas with the plasmid containing Pcin and cinR-luxI in the genome; (3) Mixing and fermenting the Halomonas obtained in step (1) (ii) and the Halomonas obtained in step (2) (ii).

5. The method for constructing an interactive stable expression population response mixed bacteria induction system according to claim 4, characterized in that: The Halomonas obtained in step (1) (ii) and the Halomonas obtained in step (2) (ii) are mixed in any volume ratio of 1:9, 1:4, 1:1, 4:1, or 9:1; The time for mixing the Halomonas obtained in (ii) of step (1) and the Halomonas obtained in (ii) of step (2) is 0h-16h.

6. An interactive stable expression population response mixed bacteria induction expression amplification system, characterized in that: The interactive stable expression population response mixed bacteria induction expression amplification system comprises the Plux-luxR-cinI induction expression system of claim 1, the Pcin-cinR-luxI induction expression system of claim 1, a T7-like promoter in Halomonas, T7 RNA polymerase, and a target gene.

7. The method for constructing the interactive stable expression population response mixed bacteria induction expression amplification system according to claim 6, characterized in that: The following steps are involved: (1) replacing the pTac-RNAP promoter on the genome of the Halomonas bacteria containing cinR-luxI in claim 4 with the wild-type promoter Pcin in claim 3 to obtain Halomonas bacteria containing the wild-type promoter Pcin in the genome; (2) replacing the pTac-RNAP promoter on the genome of Halomonas containing luxR-cinI of claim 4 with the wild-type promoter Plux of claim 2 to obtain the wild-type promoter Plux Halomonas; (3) inserting the T7-like promoter and the nucleotide sequence of the target gene into the plasmid to obtain a recombinant plasmid; (4) The recombinant plasmid of step (3) is transformed into the Halomonas obtained in step (1) and the Halomonas obtained in step (2), respectively, and mixed bacteria and fermentation culture are performed.

8. An interactive stable expression population response mixed bacteria induced expression inhibition system, characterized in that: The interactive stable expression population response mixed bacteria induced expression inhibition system comprises the Plux-luxR-cinI induced expression system according to claim 1, the Pcin-cinR-luxI induced expression system according to claim 1, dCas9, gRNA, and target gene.

9. The method for constructing the interactive stable expression population response mixed bacteria induced expression inhibition system according to claim 8, characterized in that: The following steps are involved: (1) constructing a Pcin-dCas9-promoter-target gene expression module, and inserting the expression module into a plasmid to obtain a first plasmid; Constructing a Pcin-gRNA expression module, and inserting the expression module into a plasmid to obtain a second plasmid; The first plasmid and the second plasmid are co-transformed into the Halomonas of claim 4 whose genome contains cinR-luxI to obtain Halomonas with double plasmids; (2) constructing a Plux-dCas9-promoter-target gene expression module, and inserting the expression module into the plasmid to obtain a third plasmid; Constructing a Plux-gRNA expression module, inserting the expression module into the plasmid to obtain a fourth plasmid; The third plasmid and the fourth plasmid are co-transformed into the Halomonas of claim 4 whose genome contains luxR-cinI to obtain Halomonas with double plasmids; (3) Mixing and fermenting the Halomonas with double plasmids obtained in steps (1) and (2).

10. Use of the interactive stable expression population response mixed bacteria induction system according to any one of claims 1 to 3, the interactive stable expression population response mixed bacteria induction expression amplification system according to claim 6, or the interactive stable expression population response mixed bacteria induction inhibition expression system according to claim 8 in Halomonas.

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