Microorganism self-induced expression system without resistance gene marker and construction method of microorganism self-induced expression system
By constructing plasmids carrying quorum sensing and nutrient deficiency recovery systems and integrating them onto a single plasmid, the dependence on antibiotics and inducers in microbial fermentation was solved, achieving green and environmentally friendly self-induced expression and reducing costs.
Patent Information
- Application Number
- CN202511550659.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-28
- Publication Date
- 2026-02-17
AI Technical Summary
Existing technologies rely on antibiotics and inducers for microbial fermentation, which increases application costs and environmental burden. There is a lack of organic integration schemes for maintaining stable plasmid expression without antibiotic labeling and for quorum sensing self-induced expression.
We constructed a plasmid carrying a quorum sensing system and a nutrient deficiency recovery system, and integrated them into the same plasmid using the Gibsonassembly seamless ligation method to achieve self-induced expression, avoiding the use of antibiotics and external inducers.
It enables automatic switching to functional expression in the early stages of bacterial growth without relying on antibiotics and inducers, reducing application costs and achieving green and environmentally friendly protein expression.
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Figure CN121538239A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a microbial self-induced expression system without resistance gene markers and its construction method. Background Technology
[0002] In fermentation engineering, plasmids are commonly used as gene vectors for regulation within host cells. While plasmid vectors are simple to manipulate and offer high expression levels, plasmid stability requires antibiotics and corresponding resistance genes. Antibiotic use not only increases application costs and causes environmental pollution but also leads to bacterial resistance. Furthermore, the use of resistance genes carries the risk of gene drift; unstable distribution and degradation of antibiotics in the culture medium can also result in reduced plasmid copy numbers and unstable bacterial phenotypes. In the food and pharmaceutical industries, antibiotic use can also lead to antibiotic residues in the final product. Due to the urgent need for green and biomanufacturing, bioengineering is receiving increasing attention. Antibiotic-free plasmid systems can effectively address these issues, such as auxotrophic regression, inhibitory protein titration, toxicity-antitoxicity screening, and RNA selection encoding. Auxotrophic regression involves artificially knocking out essential genes from the host cell genome and then carrying these essential genes on a plasmid to maintain the plasmid's stable presence in the host cell. This method avoids antibiotic use while retaining the advantages of high plasmid copy numbers and high expression.
[0003] Typically, engineered fermentation of E. coli uses pET plasmids as vectors for the target gene. While this vector, when combined with E. coli (BL21), can express high yields of the target protein, it requires the inducer isopropyl-β-D-thiogalactoside (IPTG) to induce expression. The use of inducers not only increases the environmental burden but also raises the application cost in engineered fermentation.
[0004] Quorum sensing is a cell-to-cell communication system in bacteria. Bacteria sense the density of their colony by secreting diffusible small molecular signals, thereby inducing the coordinated expression of a specific set of genes at the transcriptional level. Quorum sensing involves the production, release, and detection of extracellular signaling molecules within the colony; these molecules are called autoinducers. The concentration of autoinducers increases with increasing cell density. Once a certain concentration is reached, they can bind to intracellular receptor proteins to form receptor-protein complexes, ultimately altering the overall pattern of gene expression. To date, autoinducer signaling molecules have been identified into three main categories: N-acylated homoserine lactones; amino acids and short peptides, which primarily facilitate intraspecific communication; and furanoylborate diesters, which can assist in interspecific communication.
[0005] Although both types of technologies (antibiotic-free maintenance and self-induction) have made progress, existing technologies are largely isolated. Currently, there is a lack of an integrated solution that organically combines the advantages of "antibiotic-free plasmid stability maintenance" and "quorum-sensing self-inducible expression." Specifically, existing quorum-sensing expression systems typically still rely on antibiotic labels to maintain plasmid stability, failing to fundamentally eliminate antibiotic use; while conventional auxotrophic host bacteria still require external inducers to initiate expression, failing to achieve intelligent fermentation processes.
[0006] Therefore, building a truly green and intelligent protein expression platform that can simultaneously and completely eliminate the use of antibiotics and external inducers is a technical problem that needs to be solved. Summary of the Invention
[0007] One of the objectives of this invention is to provide a microbial self-induced expression system without resistance gene markers, in order to solve the problem that microbial fermentation in the prior art relies on antibiotics and inducers, which increases application costs and environmental burden.
[0008] The second objective of this invention is to provide a method for constructing a microbial self-induced expression system without resistance gene markers.
[0009] The objective of this invention can be achieved through the following technical solutions: Microbial self-induced expression systems without resistance gene markers include plasmids carrying quorum sensing systems, plasmids carrying nutrient deficiency recovery systems, and chassis microorganisms.
[0010] The plasmid carrying the quorum sensing system includes the LuxI gene, LuxR gene, promoter PL, promoter PLux, and pYYDT vector. The PL promoter initiates the tandem expression of the LuxR and LuxI genes, ultimately producing LuxR protein and LuxI enzyme. The PLux promoter initiates the expression of exogenous genes.
[0011] The nucleotide sequence of the LuxI gene is shown in SEQ ID NO. 1.
[0012] The nucleotide sequence of the LuxR gene is shown in SEQ ID NO.2.
[0013] The nucleotide sequence of the promoter PL is shown in SEQ ID NO. 3.
[0014] The nucleotide sequence of the promoter PLux is shown in SEQ ID NO.4.
[0015] The plasmid carrying the nutrient deficiency compensation system includes a nucleic acid sequence encoding diaminopimelic acid (dap), a PCN promoter, and a pYYDT vector.
[0016] The nucleic acid sequence encoding diaminopimelic acid (dap) is shown in SEQ ID NO. 5.
[0017] The nucleic acid sequence of the PCN promoter is shown in SEQ ID NO. 6.
[0018] A method for constructing a microbial self-induced expression system without resistance gene markers includes the following steps: (1) Constructing plasmids carrying quorum sensing systems; (2) Constructing plasmids carrying a nutrient deficiency compensation system; (3) The quorum sensing system and the nutrient deficiency replenishment system are integrated into the same plasmid using molecular biology methods to obtain an integrated plasmid that has both self-induction and plasmid stabilization functions. (4) Clone the exogenous gene into the integration plasmid to construct the final expression plasmid; (5) The expression plasmid is electrotransformed into the chassis microorganisms, and positive clones are verified by plate culture and molecular biology methods to obtain the self-induced expression engineered bacteria without resistance gene markers.
[0019] The exogenous gene is the nucleic acid sequence of recombinant type XVII collagen or the amino acid sequence of type III collagen.
[0020] The nucleic acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO. 7.
[0021] The amino acid sequence of the type III collagen is shown in SEQ ID NO. 8.
[0022] The chassis microorganism is Escherichia coli WM3064.
[0023] Furthermore, the plasmid carrying the quorum sensing system is specifically constructed and obtained through the following steps: S11. Using the Gibson assembly seamless ligation method, the LuxI gene, LuxR gene, promoter PL, promoter PLux and pYYDT vector backbone fragment were ligated to obtain the DNA assembly product pYYDT-LuxI-LuxR. S12. The DNA assembly product pYYDT-LuxI-LuxR was transferred into the substrate microorganisms by chemical or electrochemical transformation and plated on 50ug / mL 2×YT solid plates. It was then incubated at 37℃ for 18-24h. Single colonies were selected from the plates for PCR and verified by Sanger sequencing. Plasmids from bacterial cultures with correct sequencing results were extracted and identified by restriction endonuclease digestion to exclude false positive plasmids caused by DNA fragment duplication or incorrect ligation. The plasmid carrying the quorum sensing system was obtained and denoted as plasmid pYYDT-LuxI-LuxR.
[0024] Furthermore, the LuxI gene, LuxR gene, promoter PL, and promoter PLux in S11 were all obtained by high-fidelity PCR amplification and purification.
[0025] Furthermore, the specific operating steps for S11 are as follows: 100 ng of the pYYDT vector backbone fragment was fixed. The pYYDT vector backbone fragment, LuxI gene, LuxR gene, promoter PL and promoter PLux were mixed in equimolar amounts. Then, sterile double-distilled water was added to make up to 10 μL. The mixture was then mixed with 10 μL of Libson Mixture and incubated at 50 °C for 1 h to obtain the DNA assembly product pYYDT-LuxI-LuxR.
[0026] Furthermore, the plasmid carrying the nutrient deficiency compensation system is constructed through the following steps: S21. Using the Gibsonassembly seamless ligation method, the nucleic acid sequence encoding diaminopimelic acid (dap) and the vector pYYDT fragment containing the PCN promoter are ligated to obtain the DNA assembly product pYYDCT-dap. S22. The DNA assembly product pYYDTC-dap was transferred into the substrate microorganisms by chemical or electrochemical transformation and plated on 50ug / mL 2×YT solid plates. It was then incubated at 37℃ for 18-24h. Single colonies were selected from the plates for PCR and verified by Sanger sequencing. Plasmids from bacterial cultures with correct sequencing results were extracted and identified by restriction endonuclease digestion to exclude false positive plasmids caused by DNA fragment duplication or incorrect ligation. The plasmid carrying the nutrient deficiency compensation system was obtained and denoted as plasmid pYYDTC-dap.
[0027] Furthermore, the pYYDT vector fragment containing the PCN promoter is obtained through the following steps: The PCN promoter nucleic acid sequence was amplified and incorporated into the pYYDT vector. 100 ng of the pYYDT vector backbone fragment was immobilized. The PCN promoter and pYYDT vector were mixed equimolarly, and sterile double-distilled water was added to a final volume of 10 μL. This volume was then mixed with 10 μL of Gibson Mixture and incubated at 50 °C for 1 h to obtain a DNA assembly product containing the PCN promoter. This PCN promoter-containing DNA assembly product was then electroporated into a substrate of microorganisms and plated on 2×YT solid plates containing 50 μg / mL kanamycin. Colonies were selected for PCR and Sanger sequencing verification. Plasmids from bacteria with correct sequencing results were extracted and digested with restriction endonucleases to eliminate false-positive plasmids caused by duplicate or incorrect DNA ligation, yielding the vector pYYDT containing the PCN promoter, denoted as vector pYYDTC.
[0028] Furthermore, the specific steps for constructing a microbial self-induced expression system without resistance gene markers are as follows: (1) Amplify the PCN-dap portion of plasmid pYYDTC-dap and plasmid pYYDT-LuxI-LuxR to obtain the DNA assembly product pYYDT-LuxI-LuxR-PCN-dap; (2) The DNA assembly product pYYDT-LuxI-LuxR-PCN-dap was transferred into the substrate microorganisms by chemical or electrochemical transformation, and then plated on 50 μg / mL kanamycin 2×YT solid plates to amplify the plasmid. Colonies were selected for PCR and verified by Sanger sequencing. The bacteria with correct sequencing results were self-induced expression engineered bacteria carrying the complementation system, and the extracted plasmid was an integrated plasmid with both self-induction and plasmid stabilization functions. (3) Clone the exogenous gene into the integration plasmid to construct the final expression plasmid; (4) The final expression plasmid is electrotransformed into the chassis microorganisms, and positive clones are verified by plate culture and molecular biology methods to obtain the self-induced expression engineered bacteria without resistance gene markers.
[0029] Further, the PCN-dap portion of the plasmid pYYDTC-dap is obtained through the following steps: The dap nucleic acid sequence and the pYYDTC vector backbone were amplified. 100 ng of the pYYDTC vector backbone fragment was immobilized. The dap nucleic acid sequence and the pYYDTC vector backbone were mixed equimolarly, and sterile double-distilled water was added to a final volume of 10 μL. This was then mixed with 10 μL of Gibson Mixture and incubated at 50 °C for 1 h to obtain the DNA assembly product. The DNA assembly product was then electroporated into WM3064 competent cells and plated on 2×YT solid plates containing 50 μg / mL kanamycin. Colonies were selected for PCR and Sanger sequencing verification. Plasmids were extracted from bacteria with correct sequencing results and digested with restriction endonucleases to eliminate false-positive plasmids caused by DNA fragment duplication or incorrect ligation, yielding engineered bacteria carrying the pYYDTC-dap plasmid. The PCN-dap fraction was amplified using the pYYDTC-dap plasmid as a template.
[0030] Furthermore, the chassis microorganism is Escherichia coli WM3064.
[0031] Furthermore, the GibsonMixture preparation method is as follows: Take 160 μL of ISO Buffer (5x), add 80 μL of 40 U / μL Taq DNA ligase, 10 μL of 2 U / μL Q5 DNA polymerase, and 0.32 μL of 10 U / μL T5 exonuclease in sequence, mix well, and then add sterile double-distilled water to a final volume of 600 μL. Finally, dispense 10 μL per tube and store at -20℃ for later use.
[0032] The beneficial effects of this invention are: This invention utilizes synthetic biology methods and tools to genetically modify chassis microorganisms, successfully enabling microorganisms to grow in the early stages of microbial culture without relying on antibiotics and inducers. Once the bacterial population density reaches a certain level, it automatically switches to functional expression, realizing dynamic allocation of resources within the bacteria, truly achieving green and environmentally friendly practices while reducing application costs. Attached Figure Description
[0033] The invention will now be further described with reference to the accompanying drawings.
[0034] Figure 1 This is the plasmid map of the quorum sensing system of this invention; Figure 2 This is a plasmid map of the nutrient deficiency compensation system of this invention; Figure 3 This is the quorum sensing plasmid map of the diaminopimelic acid activator of this invention; Figure 4 This is the spectrum of the quorum sensing fluorescent expression plasmid for initiating diaminopimelic acid according to the present invention; Figure 5This is a graph showing the effect of the compensating plasmid constructed by the nutrient deficiency compensating method of this invention on the growth of the chassis strain; Figure 6 This is a flow cytometry analysis and evaluation result diagram of the integration of the quorum sensing system and the nutrient deficiency replenishment system of this invention; Figure 7 This invention utilizes fluorescence microscopy to capture images showing the strength of functional expression in a microbial self-induced expression system without resistance gene markers. Figure 8 This is a graph showing the ability of a microbial self-induced expression system without resistance gene markers to express type XVII collagen in Example 4 of this invention. Figure 9 This is a diagram showing the expression, isolation, purification, and testing of type III collagen using a microbial self-induced expression system without resistance gene markers in Example 5 of this invention. Figure 10 This is an SDS-PAGE polyacrylamide gel electrophoresis image comparing the antibiotic-free quorum sensing system of this invention with the traditional "antibiotic labeling + IPTG induction" system and the antibiotic-dependent self-induced expression system. Figure 11 This figure shows the test results of the expression of the target protein by the quorum sensing system of the present invention without the addition of antibiotics, compared with the traditional "antibiotic labeling + IPTG induction" system and the antibiotic-dependent self-inducible expression system. Detailed Implementation
[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0036] The Gibson Mixture preparation method in this application is as follows: Take 160 μL of ISO Buffer (5x), add 80 μL of 40 U / μL Taq DNA ligase, 10 μL of 2 U / μL Q5 DNA polymerase, and 0.32 μL of 10 U / μL T5 exonuclease in sequence, mix well, and then add sterile double-distilled water to make up to 600 μL. Finally, dispense into 10 μL tubes and store at -20℃ for later use. The 2×YT liquid medium formula is: 16 g / L tryptone, 10 g / L yeast extract and 5 g / L sodium chloride. The 2×YT solid medium is obtained by adding 1.5% Agar to the 2×YT liquid medium.
[0037] The present application will now be described in detail with reference to specific embodiments.
[0038] Example 1: Plasmids carrying a quorum sensing system
[0039] The plasmid carrying the quorum sensing system is obtained through the following steps: S11, Reference Figure 1 As shown, according to the pYYDT vector backbone fragment 100ng, the pYYDT vector backbone fragment, LuxI gene (as shown in SEQ ID NO. 1), LuxR gene (as shown in SEQ ID NO. 2), promoter PL (as shown in SEQ ID NO. 3) and promoter PLux (as shown in SEQ ID NO. 4) were mixed in equimolar amounts, then sterile double-distilled water was added to make up to 10 μL, and then mixed with 10 μL Gibson Mixture. The mixture was incubated at 50℃ for 1 h to obtain the DNA assembly product pYYDT-LuxI-LuxR. S12. The DNA assembly product pYYDT-LuxI-LuxR was transferred into the chassis microorganism EcoliWM3064 by electroporation, and then plated on a 50ug / mL kanamycin 2×YT solid plate and incubated at 37℃ for 18h. Single colonies were selected from the plate for PCR and verified by Sanger sequencing. Plasmids from bacterial cultures with correct sequencing results were extracted and identified by restriction endonuclease digestion to exclude false positive plasmids caused by DNA fragment duplication or incorrect ligation. The plasmid carrying the quorum sensing system was obtained and denoted as plasmid pYYDT-LuxI-LuxR.
[0040] The specific steps for transferring the DNA assembly product pYYDT-LuxI-LuxR into the chassis microorganism Ecoli WM3064 via electroporation are as follows: Take 50 μL of Ecoli WM3064 bacterial culture from a -80℃ freezer and place it on ice. Freeze-thaw for 10 min. Add 800 ng of plasmid pYYDT-LuxI-LuxR to the Ecoli WM3064 bacterial culture. After three pipetting cycles, transfer it to an electroporation cuvette. Incubate on ice for 3 min. Place the electroporation cuvette in an electroporator, set to manual mode, 2.5 kV, and click Pulse. Then add 1 mL of 2×YT liquid medium. Invert the cuvette 5 times and transfer it to a sterile 1.5 mL EP tube. Incubate at 37℃ and shake at 200 rpm for 1 h to obtain the recovery solution. Spread the recovery solution onto a 2×YT solid plate containing 50 μg / mL kanamycin and incubate at 37℃ for 24 h to complete the transformation process.
[0041] The preparation process of Ecoli WM3064 bacterial culture is as follows: Ecoli WM3064 culture preserved at -80℃ was streaked onto 2×YT agar plates and incubated overnight at 37℃ with the plates inverted. Single colonies were picked and inoculated into 3 mL of 2×YT liquid medium. The culture was then incubated overnight at 37℃ with shaking at 200 rpm. The above seed culture was then transferred to fresh 2×YT liquid medium at a 1% inoculation rate and incubated at 37℃ with shaking at 150 rpm for approximately 2 hours. OD of the bacterial culture was then monitored. 600 Value. When OD 600 When the bacterial concentration reaches 0.48-0.55, immediately incubate the bacterial culture in an ice-water bath for 20 minutes, mixing every 5 minutes. Aliquot the bacterial culture into pre-chilled 50mL centrifuge tubes and centrifuge at 4℃ and 4000 rpm for 15 minutes, discarding the supernatant. Resuspend the bacterial cells in an equal volume of pre-chilled double-distilled water, centrifuge under the same conditions, and discard the supernatant. Resuspend the bacterial cells again in an equal volume of pre-chilled 10% glycerol, centrifuge under the same conditions, discard the supernatant, and collect the bacterial cells. Gently resuspend the bacterial cells in 0.8% of the original culture volume of pre-chilled 10% glycerol, aliquot 50μL / tube into pre-chilled 1.5mL EP tubes, and immediately store at -80℃ for later use.
[0042] Example 2: Construction of a plasmid carrying a nutrient deficiency compensation system
[0043] Plasmids carrying nutrient deficiency compensation systems are obtained through the following steps: S21, Reference Figure 2 As shown, the nucleic acid sequence encoding diaminopimelic acid (dap) (as shown in SEQ ID NO. 5) and the pYYDT vector backbone fragment containing the PCN promoter were ligated using the Gibson assembly seamless ligation method. 100 ng of the pYYDT vector backbone fragment containing the PCN promoter was mixed equimolarly with the dap fragment, and sterile double-distilled water was added to a final volume of 10 μL. This was then mixed with 10 μL of Gibson Mixture and incubated at 50 °C for 1 h to obtain the DNA assembly product pYYDTC-dap. S22. The DNA assembly product pYYDTC-dap was transferred into the chassis microorganisms by electroporation (the electroporation method is the same as in Example 1, only the DNA assembly product is different). Then, it was plated on a 50ug / mL 2×YT solid plate and incubated at 37℃ for 24h. Single colonies were selected from the plate for PCR and verified by Sanger sequencing. The engineered bacteria WM3064 / PCN-dap with correct sequencing results were retained. Plasmids from the bacterial cultures with correct sequencing results were extracted and identified by restriction endonuclease digestion to exclude false positive plasmids caused by DNA fragment duplication or incorrect ligation. The complement plasmid carrying the initiator diaminopimelic acid was obtained and denoted as plasmid pYYDTC-dap.
[0044] The pYYDT vector fragment containing the PCN promoter is obtained through the following steps: The PCN promoter nucleic acid sequence was amplified and incorporated into the pYYDT vector. 100 ng of the pYYDT vector backbone fragment was immobilized. The PCN promoter and pYYDT vector were mixed equimolarly, and sterile double-distilled water was added to a final volume of 10 μL. This was then mixed with 10 μL of Gibson Mixture and incubated at 50 °C for 1 h to obtain a DNA assembly product containing the PCN promoter. This PCN promoter-containing DNA assembly product was then electroporated into WM3064 competent cells and plated on 2×YT solid plates containing 50 μg / mL kanamycin. Colonies were selected for PCR and Sanger sequencing verification. Plasmids from bacteria with correct sequencing results were extracted and digested with restriction endonucleases to eliminate false-positive plasmids caused by DNA fragment duplication or incorrect ligation, yielding the PCN promoter-containing vector pYYDT, denoted as vector pYYDTC.
[0045] The engineered strain WM3064 / PCN-dap obtained from S22 and strain WM3064 were inoculated at 1% in 5 mL of 2×YT liquid medium. For strain WM3064, an additional 50 μg / mL of 2,6-diaminopimelic acid was added during culturing. 100 μL of each bacterial culture was spread onto 2×YT solid agarose plates containing 50 μg / mL kanamycin. Both cultures were incubated at 37℃ for 48 h. The effect of the compensating plasmid constructed using the auxotrophic replacement method of this application on the growth of the chassis strain was observed. The results are as follows: Figure 5 As shown, by Figure 5 It can be seen that the chassis microbial strain WM3064 can express the growth essential diaminopimelic acid using the constructed gene complementation plasmid pYYDTC-dap.
[0046] Example 3: Integration of quorum sensing system with nutrient deficiency recovery system
[0047] The quorum sensing system was integrated with the nutrient deficiency replenishment system to obtain a recombinant plasmid, which was obtained through the following steps: Step (1), refer to Figure 3 As shown, 100 ng of plasmid pYYDT-LuxI-LuxR fragment from Example 1 was fixed. The PCN-dap portion of plasmid pYYDTC-dap from Example 2 was mixed with the plasmid pYYDT-LuxI-LuxR fragment in equimolar amounts. Then, sterile double-distilled water was added to make up to 10 μL, and then mixed with 10 μL of Gibson Mixture. The mixture was incubated at 50 °C for 1 h to obtain the DNA assembly product pYYDT-LuxI-LuxR-PCN-dap. The PCN-dap portion of plasmid pYYDT-dap is obtained through the following steps: The dap nucleic acid sequence and the pYYDTC vector backbone were amplified. 100 ng of the pYYDTC vector backbone fragment was immobilized. The dap nucleic acid sequence and the pYYDTC vector backbone were mixed equimolarly, and sterile double-distilled water was added to a final volume of 10 μL. This was then mixed with 10 μL of Gibson Mixture and incubated at 50 °C for 1 h to obtain the DNA assembly product. The DNA assembly product was then electroporated into WM3064 competent cells and plated on 2×YT solid plates containing 50 μg / mL kanamycin. Colonies were selected for PCR and Sanger sequencing verification. Plasmids were extracted from bacteria with correct sequencing results and digested with restriction endonucleases to eliminate false-positive plasmids caused by DNA fragment duplication or incorrect ligation, yielding engineered bacteria carrying the pYYDTC-dap plasmid. The extracted plasmid was then amplified by high-fidelity PCR and purified by agarose gel electrophoresis to obtain the PCN-dap fraction.
[0048] Step (2): The DNA assembly product pYYDT-LuxI-LuxR-PCN-dap was transferred into the substrate microorganisms by electroporation (the electroporation method is the same as in Example 1, only the DNA assembly product is different). The product was then plated on a 50 μg / mL kanamycin 2×YT solid plate for plasmid amplification. Colonies were selected for PCR and verified using Sanger sequencing. Bacteria with correct sequencing results were self-induced expression engineered bacteria carrying an complementation system. The plasmid was extracted from the bacterial culture with correct sequencing results and designated as plasmid pYYDT-LuxI-LuxR-PCN-dap. Step (3), refer to Figure 4 The contents shown are as follows: the exogenous fluorescent protein gene sfGFP (nucleic acid sequence as shown in SEQ ID NO. 9) and the plasmid pYYDT-LuxI-LuxR-PCN-dap backbone are amplified. 100 ng of the pYYDT-LuxI-LuxR-PCN-dap backbone fragment is mixed with an equimolar amount of the sfGFP fragment, and sterile double-distilled water is added to make up to 10 μL. Then, it is mixed with 10 μL of Gibson Mixture and incubated at 50℃ for 1 h to obtain the DNA assembly product pYPSD-PCN-dap-sfGFP plasmid. The exogenous sfGFP gene is then promoted using the Plux promoter. Step (4): The DNA assembly product pYPSD-PCN-dap-sfGFP plasmid was transferred into the chassis microorganisms by electroporation (the electroporation method is the same as in Example 1, only the DNA assembly product is different). Then, it was plated on a 50 μg / mL kanamycin 2×YT solid plate for culture to amplify the plasmid. Colonies were selected for PCR and verified by Sanger sequencing. Plasmids were extracted from bacteria with correct sequencing results and digested with restriction endonucleases to eliminate false positive plasmids caused by DNA fragment duplication or incorrect ligation, resulting in fluorescent engineered bacteria carrying a complement plasmid for initiating diaminopimelic acid expression. Step (5): Inoculate the fluorescent engineered bacteria into 10 mL of 2×YT liquid medium and incubate at 37℃ and 200 rpm. At 3h, 6h, 12h, 24h, 36h, and 48h, take 1 mL of the bacterial culture, centrifuge at 12000g for 2 min, discard the supernatant, resuspend in 1 mL of deionized water, and add to a flow cytometry tube. Detect the fluorescence expression and distribution at each time point using flow cytometry. Figure 6 As shown; the fluorescent engineered bacteria were inoculated into 10 mL of 2×YT liquid medium and cultured at 37℃ and 200 rpm. After 48 h, 1 mL of the bacterial suspension was taken, centrifuged at 12000g for 2 min, the supernatant was discarded, and the suspension was resuspended in 1 mL of deionized water. The fluorescence expression was then photographed using a fluorescence microscope. Figure 7 As shown; by Figure 6 and Figure 7 It can be seen that the system has a strong expressive effect.
[0049] Example 4
[0050] A method for constructing a microbial self-induced expression system without resistance gene markers includes the following steps: Step (1): The nucleic acid sequence of recombinant type XVII collagen (denoted as COL17A1, as shown in SEQ ID NO.7) is assembled into the plasmid pYYDT-LuxI-LuxR-PCN-dap obtained in Example 3 to obtain plasmid pYPSD-COL17A1-PCN-dap; Specifically, the nucleic acid sequence COL17A1 of type XVII collagen was amplified and the pYYDT-LuxI-LuxR-PCN-dap backbone was mixed with 100 ng of the pYYDT-LuxI-LuxR-PCN-dap backbone in an equimolar mixture with the COL17A1 fragment. Sterile double-distilled water was added to bring the volume to 10 μL, and then mixed with 10 μL of Gibson Mixture. The mixture was incubated at 50 °C for 1 h to obtain the plasmid DNA assembly product pYPSD-COL17A1-PCN-dap. The exogenous gene for type XVII collagen was then started using the Plux promoter. The plasmid pYPSD-COL17A1-PCN-dap was electrotransformed into Escherichia coli WM3064 (the electrotransformation method was the same as in Example 1). The plasmid was spread on 2×YT plates containing 50 μg / mL kanamycin resistance solid. Positive transformants were screened and sequenced and enzyme digested for verification. Single colonies on the plates were picked to obtain the expression strain. Step (2): Inoculate the expression strain into 2×YT medium and culture at 37℃ and 200rpm for 24h to obtain seed liquid. Inoculate 1% of the seed liquid into 100mL of 2×YT medium and ferment at 37℃ and 200rpm for 48h. Centrifuge the fermentation broth to collect the bacterial sludge. After washing with deionized water, set the ultrasonic power to 300W, turn on for 3s and turn off for 5s, for a total duration of 15min to obtain the broken bacterial sample solution. Step (3): Take the broken bacterial cell sample solution for SDS-PAGE detection; Test results are as follows Figure 8 As shown, Figure 8 The first column on the left is the marker, the second column is the original recombinant type XVII collagen sample, and the third column is the recombinant type XVII collagen sample after the original solution was diluted 5 times.
[0051] The results showed that the designed fermentation system could effectively express exogenous recombinant proteins. The protein content was determined by the Coomassie brilliant blue method and calculated using ImageJ grayscale scanning to be 0.53 mg / mL of the bacterial culture.
[0052] Example 5
[0053] A method for constructing a microbial self-induced expression system without resistance gene markers includes the following steps: Step (1): The amino acid sequence of type III collagen (denoted as COL3A1, as shown in SEQ ID NO.8) is assembled into the plasmid pYYDT-LuxI-LuxR-PCN-dap obtained in Example 3 to obtain the pYPSD2-COL3A1-PCN-dap plasmid. Specifically, the nucleic acid sequence COL3A1 of type III collagen was amplified and the pYYDT-LuxI-LuxR-PCN-dap backbone was mixed with 100 ng of the pYYDT-LuxI-LuxR-PCN-dap backbone in an equimolar mixture with the COL3A1 fragment. Sterile double-distilled water was added to bring the volume to 10 μL, and then mixed with 10 μL of Gibson Mixture. The mixture was incubated at 50 °C for 1 h to obtain the plasmid pYPSD-COL3A1-PCN-dap. The exogenous gene for type III collagen was then started using the Plux promoter. The plasmid pYPSD-COL3A1-PCN-dap was electrotransformed into Escherichia coli WM3064 (the electrotransformation method is the same as in Example 1). The plasmid was spread on 2×YT plates containing 50 μg / mL kanamycin resistance solid. Positive transformants were screened and sequenced and enzyme digested for verification. Single colonies on the plates were picked to obtain the expression strain. Step (2): Inoculate the expression strain into 2×YT medium and culture at 37℃ and 200rpm for 24h to obtain seed liquid. Inoculate 1% of the seed liquid into 100mL of 2×YT medium and ferment at 37℃ and 200rpm for 48h. Centrifuge the fermentation broth to collect the bacterial sludge. After washing with deionized water, set the ultrasonic power to 300W, turn on for 3s and turn off for 5s, for a total duration of 15min to obtain the broken bacterial sample. Step (3): Since a 6×His tag is inserted at the N-terminus of the protein, Ni column affinity chromatography can be used to purify the lysed bacterial cell sample solution. Take the lysed bacterial cell sample solution onto the Ni column and use imidazole for gradient elution. Take the elution solution for SDS-PAGE detection. Test results are as follows Figure 9 As shown, Figure 9 The first column on the left is the marker, the second column is the stock solution of type III collagen, the third column is the flow-through solution, the fourth column is elution with 20 mM imidazole, the fifth column is elution with 40 mM imidazole, the sixth column is elution with 60 mM imidazole, and the seventh column is elution with 100 mM imidazole. Experimental results show that the designed system can effectively ferment and express tandem type III collagen, and the protein purity reaches over 70% after preliminary purification by Ni column affinity chromatography.
[0054] Comparative Example 1
[0055] The construction of expression strains based on pET-28 is as follows: The nucleic acid sequence of recombinant type XVII collagen (as shown in SEQ ID NO. 7) was assembled into the pET-28(a) vector using the Gibson method to obtain a recombinant plasmid. The recombinant plasmid was electrotransformed into Escherichia coli BL21(DE3), and then plated on 2×YT plates containing 50 μg / mL kanamycin resistance solid. Positive transformants were screened and sequenced and enzyme digested for verification. Single colonies on the plates were picked to obtain the expression strain.
[0056] Comparative Example 2
[0057] The effect of the pET system on expressing type XVII collagen was examined under different culture conditions. The specific methods are as follows: (1) Antibiotic-free quorum sensing system culture method (PSD-PCN-dap system): The expression strain obtained in Example 4 was inoculated at 1% into 100 mL of 2×YT liquid medium and cultured at 37°C and 200 rpm for 48 h. (2) “Antibiotic labeling + IPTG induction” system culture method (pET system): The expression strain obtained in Comparative Example 1 was inoculated at 1% into 100mL of 2×YT liquid medium containing 50μg / mL kanamycin and cultured at 37℃ and 200rpm until the OD value reached 0.6-0.8. Then, IPTG inducer with a final concentration of 0.4mM was added and cultured for 48h. (3) Antibiotic-dependent self-induced expression system (PSD system) culture method: The expression strain obtained in Example 4 was inoculated at 1% into 100mL of 2×YT liquid medium containing 50μg / mL kanamycin and cultured at 37℃ and 200rpm for 48h; The fermentation broths of the three types of bacterial cultures were centrifuged to collect bacterial sludge. After washing with deionized water, the ultrasonic power was set to 300W, with the ultrasonic cycle on for 3 seconds and off for 5 seconds, for a total duration of 15 minutes. The resulting fragmented bacterial samples were then analyzed by SDS-PAGE. Figure 10 As shown, the leftmost and rightmost lanes are protein markers, lane 2 from the left is for pET system protein expression, lane 3 from the left is for PSD-PCN-dap system protein expression, and lane 4 from the left is for PSD system protein expression. The content of the target protein was calculated using the Coomassie Brilliant Blue assay and Image J grayscale scanning. (Details are as follows...) Figure 11 As shown.
[0058] Depend on Figure 10 and Figure 11 It can be seen that the protein expression level of the pET system is 0.64-0.66 mg / mL; the protein expression level of the PSD system is 0.57-0.59 mg / mL; and the protein expression level of the PSD-PCN-dap system is 0.53-0.54 mg / mL.
[0059] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A microbial autoinduction expression system without a resistance gene marker, characterized in that, The plasmid carrying a quorum sensing system, a plasmid carrying a nutritional deficiency complementation system and a chassis microorganism.
2. The antibiotic-free marker gene free microbial auto-induction expression system according to claim 1, wherein, The plasmid carrying a quorum sensing system comprises a LuxI gene, a LuxR gene, a promoter PL, a promoter PLux and a pYYDT vector.
3. The antibiotic-free marker gene free microbial auto-induction expression system according to claim 2, wherein, The nucleotide sequence of the LuxI gene is shown in SEQ ID NO. 1, the nucleotide sequence of the LuxR gene is shown in SEQ ID NO. 2, the nucleotide sequence of the promoter PL is shown in SEQ ID NO. 3, and the nucleotide sequence of the promoter PLux is shown in SEQ ID NO.
4.
4. The antibiotic-free marker gene tagged microbial auto-induction expression system according to claim 1, wherein, The plasmid carrying a nutritional deficiency complementation system comprises a nucleic acid sequence encoding diaminopimelic acid, a PCN promoter and a pYYDT vector.
5. The antibiotic-free marker gene free microbial auto-induction expression system according to claim 4, wherein, The nucleic acid sequence encoding diaminopimelic acid is shown in SEQ ID NO. 5, and the nucleic acid sequence of the PCN promoter is shown in SEQ ID NO.
6.
6. A method for constructing an autoinduction expression system of a microorganism without a resistance gene marker, characterized by, A method for constructing the microorganism self-induction expression system without resistance gene markers according to any one of claims 1-5 comprises the following steps: (1) constructing a plasmid carrying a quorum sensing system; (2) constructing a plasmid carrying a nutritional deficiency complementation system; (3) integrating the quorum sensing system and the nutritional deficiency complementation system into the same plasmid by a molecular biology method to obtain an integrated plasmid having both self-induction and plasmid stability functions; (4) cloning an exogenous gene into the integrated plasmid to construct a final expression plasmid; (5) electrically transforming the expression plasmid into a chassis microorganism, verifying a positive clone by plate culture and a molecular biology method, and obtaining the self-induction expression engineering bacteria without resistance gene markers.
7. The method for constructing an antibiotic marker-free microbial auto-inducible expression system according to claim 6, wherein, The exogenous gene is a nucleic acid sequence of a recombinant type XI collagen or an amino acid sequence of a type III collagen.
8. The method for constructing an antibiotic marker-free microbial auto-induction expression system according to claim 7, wherein, The nucleic acid sequence of the recombinant type XI collagen is shown in SEQ ID NO. 7, and the amino acid sequence of the type III collagen is shown in SEQ ID NO.
8.
9. The method for constructing an antibiotic marker-free microbial auto-inducible expression system according to claim 6, wherein, The chassis microorganism is Escherichia coli WM3064.
10. The method for constructing an antibiotic marker-free microbial auto-inducible expression system according to claim 6, wherein, The plasmid carrying a quorum sensing system is obtained by the following steps: S11, using a Gibson assembly seamless ligation method to ligate a LuxI gene, a LuxR gene, a promoter PL, a promoter PLux and a pYYDT vector skeleton fragment to obtain a DNA assembly product pYYDT-LuxI-LuxR; S12, transforming the DNA assembly product pYYDT-LuxI-LuxR into a chassis microorganism by a chemical transformation or an electrical transformation method, coating on a 50ug / mL 2×YT solid plate and culturing at 37℃ for 18-24h; selecting a single colony from the plate for PCR and verifying by Sanger sequencing, extracting the plasmid in the bacterial culture with correct sequencing results, and performing enzyme digestion identification by a restriction endonuclease to exclude false positive plasmids caused by repeated ligation or incorrect ligation of DNA fragments, to obtain the plasmid carrying a quorum sensing system.