Rapid, simple and convenient transformation method applied to moraxella catarrhalis

Abstract

The invention discloses a rapid and simple transformation method applied to moraxella catarrhalis, which comprises the following steps: uniformly mixing a carrier carrying a target gene with a bacterial colony of moraxella catarrhalis, and then incubating at 32-39 DEG C for 2-5 hours; the moraxella catarrhalis is in an exponential growth phase, the OD value of a bacterial colony is 0.8-1.2, and the effective viable count is 0.5 * 10 < 8 > cfu / ml to 3 * 10 < 8 > cfu / ml. According to the rapid, simple and convenient transformation method applied to the moraxella catarrhalis, electric transformation and special instruments and equipment are not needed, the self-transformation characteristic of the moraxella catarrhalis is utilized, a carrier and thalli are directly mixed and incubated for 3-4 h, and the transformation method can be completed through conventional screening. Compared with the prior art, the method is low in experimental condition requirement, simple to operate and high in conversion success rate, and the conversion rate can reach 80% or above.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology and microbiology, and in particular to a rapid and simple transformation method applied to Moraxella catarrhalis. Background Art

[0002] Moraxella catarrhalis is the second most important pathogen causing acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in adults, second only to Haemophilus influenzae. Further research on the function and mechanism of action of Moraxella catarrhalis is important.

[0003] Gene transformation experiments are a common strategy in biological research. Currently, there are few reports on gene transformation strains of Moraxella catarrhalis. A study conducted a comprehensive evaluation of 19 trimeric substrate proteins of 15 ABC transporter systems in the genome of Moraxella catarrhalis by constructing gene knockout mutants, and studied their role in experiments evaluating infection mechanisms. The specific method is: amplify the mutant fragments by PCR and fusion PCR, and the mutant plasmid contains 3 overlapping fragments, including a fragment of about 1kb upstream of the knocked-out gene (fragment 1), a non-polar kanamycin resistance gene amplified from plasmid pUC18K (fragment 2) and a fragment of about 1kb downstream of the gene (fragment 3). The plasmid composed of fragments 1, 2, and 3 was used to transform the strain O35E, screened on a BHI plate containing 50μg / mL kanamycin, and constructed mutants. The insertion site and surrounding sequences were analyzed by sequence to confirm whether the mutant was successfully constructed. However, the method for preparing the competent cells of Moraxella catarrhalis selected was not clearly stated in this study, which is not enough for subsequent researchers to learn from its method, and this method is difficult to promote.

[0004] At the same time, in order to study the function of the outer membrane protein (OMPs) of Moraxella catarrhalis, a team used PCR with B1 and B2 primers to amplify the copB gene from the genomic DNA of Moraxella catarrhalis ATCC25240 and 8184. The 2.2kb DNA fragment was cloned into the plasmid vector pGEX 4T3. The cloned gene was expressed in Escherichia coli JM109. The research team then transformed the copB gene into the genome of Moraxella catarrhalis, but the transformation method they used was very complicated: they collected the Moraxella catarrhalis precipitate and resuspended it in SDS-PAGE sample buffer, the formula of which was: 0.12M Tris, 2.4% SDS, 2% mercaptoethanol, 23.8% glycerol, 0.006% bromophenol blue, where the SDS solution can be used as an anionic surfactant, Tris promotes the rupture of the cell membrane of Moraxella catarrhalis, glycerol can protect the cell morphology, and bromophenol blue is used for color development and acts as an indicator, followed by boiling for 10 minutes to promote the opening of ion channels. Although they successfully transferred the constructed plasmid into Moraxella catarrhalis in this study, the process of preparing competent Moraxella catarrhalis is very complicated and has a high failure rate. Tris, 2.4% SDS, and 2% mercaptoethanol may damage the cell wall and cleave the nucleic acid-protein complex. 23.8% glycerol is not sufficient to play a protective role and will affect the physiological and biochemical functions of Moraxella catarrhalis, thereby affecting the subsequent research results on the function and mechanism of Moraxella catarrhalis.

[0005] It can be seen that the existing genetic transformation technologies for Moraxella catarrhalis are complicated to operate, have high experimental requirements, and are costly, making them difficult to be widely used. In view of these problems, the present invention provides a simpler, less demanding, and more successful transformation method that can be applied to Moraxella catarrhalis, which will play an important role in promoting the study of the molecular mechanism of Moraxella catarrhalis. Summary of the invention

[0006] The existing transformation methods for Moraxella catarrhalis are relatively complicated, with a high failure rate, and conventional transformation methods are not suitable for Moraxella catarrhalis. The inventors of the present invention have tried to use electroporation for transformation and found that the efficiency is extremely low. In view of the above situation, the inventors of the present invention have developed a simple and efficient transformation method that does not require the preparation of competent cells and does not rely on special instruments through a large number of experiments and taking advantage of the certain self-transformation characteristics of Moraxella catarrhalis.

[0007] The technical solution provided by the present invention is as follows:

[0008] The first aspect of the present invention provides a rapid and convenient transformation method for Moraxella catarrhalis, wherein a vector carrying a target gene or a ligation system containing a vector carrying a target gene is evenly mixed with a colony of Moraxella catarrhalis, and then incubated at 32-39° C. for 2-5 hours;

[0009] The catarrhalis bacteria were in the exponential growth phase, the OD value of the colony was between 0.8 and 1.2, and the effective viable count was 0.5×10 8 cfu / ml-3×10 8 cfu / ml.

[0010] Preferably, the target gene includes at least one of the mutant gene fragments of uspA1 and uspA2.

[0011] In the present invention, the terms "plasmid" and "plasmid vector" are used interchangeably.

[0012] Preferably, the vector comprises a pUC plasmid vector.

[0013] Preferably, the vector carrying the target gene or the ligation system comprising the vector carrying the target gene is evenly mixed with the colony of Moraxella catarrhalis and then incubated at 37-38° C. for 3-4 h.

[0014] Preferably, the concentration of the vector carrying the target gene is 0.8-2 μg / μL.

[0015] In some preferred embodiments, when two or more mutant gene fragments are included at the same time, such as when two mutant gene fragments, uspA1 and uspA2, are included, the concentrations of the vectors carrying different target genes are 0.8-2 μg / μL, respectively.

[0016] Preferably, the rapid and convenient transformation method applied to Moraxella catarrhalis comprises the following steps:

[0017] (1) Design and synthesize the target mutant gene as an amplification template;

[0018] (2) amplifying the target mutant gene;

[0019] (3) connecting the target mutant gene to a vector to construct the vector carrying the target mutant gene;

[0020] (4) picking a single colony of Moraxella catarrhalis that has been incubated overnight for 12-24 hours, inoculating it into a bacterial enrichment medium, wherein the OD value of the Moraxella catarrhalis colony is between 0.8 and 1.2, and placing it in an air incubator at 35-39° C. for 10-30 hours;

[0021] (5) taking 5-25 μL of the constructed and extracted 0.8-2 μg / μL vector and dropping it in the central area of ​​the enrichment medium; the vector contains a selection tag;

[0022] In the present invention, the selection tag may be any tag that can screen out the vector, such as a resistance tag (also known as an antibiotic resistance gene).

[0023] (6) Pick 2-15 colonies of Moraxella catarrhalis on the enrichment medium and mix them evenly with the vector carrying the target mutant gene until a biofilm with a diameter of 1-3 cm is formed; wherein the Moraxella catarrhalis is in the exponential growth phase, the OD value of the colony is between 0.8 and 1.2, and the effective viable count is 0.5×10 8 cfu / ml-3×10 8 cfu / ml;

[0024] (7) After the biofilm is dried at room temperature, the enrichment culture medium is placed in an air incubator at 32-39° C. and cultured for 2-5 hours;

[0025] (8) Then, growing colonies were observed, the OD value of the colonies was between 0.8 and 1.2, and the effective viable count was 0.5×10 8 cfu / ml-3×10 8 cfu / ml;

[0026] Scrape off any colonies and spread them on medium containing the screening substance corresponding to the screening label;

[0027] (9) Place the culture medium containing the screening substance corresponding to the screening tag described in step (8) in an air incubator at 32-39°C for overnight culture for 12-24 hours.

[0028] Optionally, in step (3), screening and sequencing are also included to identify and construct the vector carrying the target mutant gene.

[0029] Optionally, in step (9), the following steps are also included:

[0030] Observe the colonies grown on the culture medium containing the screening substance corresponding to the screening tag, pick a single colony for passaging, and then sequence and verify;

[0031] The colonies were counted and the transformation efficiency was calculated.

[0032] Optionally, the rapid and convenient transformation method applied to Moraxella catarrhalis further comprises the following steps:

[0033] For the target mutant gene, primers are designed for amplification and sequencing to verify whether the knockout is successful on the chromosome of Moraxella catarrhalis.

[0034] More preferably, the rapid and convenient transformation method applied to Moraxella catarrhalis comprises the following steps:

[0035] (1) Design and synthesize the target mutant genes of uspA1 and uspA2 as templates for PCR amplification;

[0036] (2) designing and synthesizing upstream and downstream primers of the target mutant genes uspA1 and uspA2, introducing the upstream and downstream primers into the EcoRI / BamHI cloning site, and amplifying the target mutant genes uspA1 and uspA2 by PCR;

[0037] (3) After double digestion of the uspA1 and uspA2 target mutant genes and the pUC57 plasmid vector with EcoRI / BamHI, the target mutant genes were connected to the pUC57 plasmid vector to construct a plasmid vector carrying the uspA1 and uspA2 target mutant genes; screening and sequencing to identify the constructed plasmid vector carrying the uspA1 and uspA2 target mutant genes;

[0038] (4) picking a single colony of Moraxella catarrhalis that has been incubated overnight for 12 hours, inoculating it into a bacterial enrichment medium, wherein the OD value of the Moraxella catarrhalis colony is between 0.8 and 1.0, and placing it in an air incubator at 37° C. for 16 to 20 hours;

[0039] (5) taking 10-20 μL of the constructed and extracted 1 μg / μL plasmid vector and dropping it in the central area of ​​the enrichment medium; the vector contains a resistance tag;

[0040] (6) Pick 5-10 Moraxella catarrhalis colonies incubated for 16-20 hours on the enrichment medium and mix them with the plasmid vector carrying the target mutant genes of uspA1 and uspA2 until a biofilm with a diameter of 2 cm is formed; wherein the Moraxella catarrhalis is in the exponential growth phase, the OD value of the colony is between 0.8 and 1.0, and the effective viable count is 0.5×10 8 cfu / ml-1.5×10 8 cfu / ml;

[0041] (7) After the biofilm is dried at room temperature, the enrichment culture medium is placed in an air incubator at 37° C. and cultured for 3-4 hours;

[0042] (8) Then, growing colonies were visible to the naked eye, with an OD value of 0.8-1.0 and an effective viable count of 0.5×10 8 cfu / ml-1.5×10 8 cfu / ml;

[0043] Scrape off the colony and apply it on LB solid medium containing a resistance substance corresponding to the resistance tag;

[0044] (9) inverting the LB solid medium and placing it in an air incubator at 37° C. for overnight culture for 15 h;

[0045] Observe the colonies grown on the LB solid medium plate, pick a single colony for passage, and then sequence and verify;

[0046] The colonies were counted and the transformation efficiency was calculated;

[0047] (10) Design primers for PCR amplification and Sanger sequencing of the target mutant gene to verify whether the knockout is successful on the chromosome of Moraxella catarrhalis; if the sequencing result is consistent with the reference sequence, it means that the knockout is successful.

[0048] The second aspect of the present invention provides a Moraxella catarrhalis gene transformant, which is constructed by the above-mentioned rapid and convenient transformation method applied to Moraxella catarrhalis.

[0049] The beneficial effects of the present invention include at least:

[0050] The present invention utilizes the certain self-transformation characteristics of Moraxella catarrhalis to develop a simple and efficient transformation method that does not require the preparation of competent cells and does not rely on special instruments. Among the existing transformation methods, the CaCl2 method invented by Cohen in 1972 is simple and easy, but the transformation efficiency is low. It is only used in routine molecular biology experiments and cannot meet the requirements of DNA library construction and transformation of large fragments of DNA; the super-competent cells prepared by the Rocla / Kell method have high transformation efficiency, but require a special reagent RbCl, which limits the promotion of this method; the Hanahan method has high requirements on the operating skills and reagents of the experimenter, but the repeatability is not high; the Inoue method proposed by Inoue H. et al. in 1990 has a high transformation efficiency, but has high requirements on the OD600 value of bacteria, and the culture temperature must be controlled at 18°C, the culture time is as long as 20-50h, and the operation process is relatively cumbersome; some laboratories use the modified TFB or FB method, which is to add a lot of trace ions to improve the conversion rate, but the buffer preparation is cumbersome and costly, and it is difficult to be widely adopted; the electroporation method relies on a short electric shock to promote DNA to enter the bacteria, and has a high conversion rate, but the electroporation method requires special instruments and equipment such as electroporators, and general laboratories do not have the conditions to carry out electroporation experiments. Moreover, in the present invention, neither the electroporation method nor the heat shock method can transfer DNA into Moraxella catarrhalis, and the membrane protein cannot be transferred by the conventional electroporation method or the calcium chloride method, which may destroy the membrane protein and is not conducive to the self-transformation characteristics of this bacterium.

[0051] In the existing scheme, competent Moraxella catarrhalis is prepared by preparing a complex buffer with multiple components, but the preparation process is very complicated and has a high failure rate. Tris, 2.4% SDS, and 2% mercaptoethanol may damage the cell wall and lyse the nucleic acid-protein complex, and 23.8% glycerol is not enough to protect it, which will affect the physiological and biochemical functions of Moraxella catarrhalis, thereby affecting the subsequent research results on the function and mechanism of Moraxella catarrhalis. The method of the present invention is not only simple to operate and has a high success rate, but also does not have other chemical substances that affect the normal function of Moraxella catarrhalis, which facilitates the subsequent research on the function and mechanism of Moraxella catarrhalis.

[0052] The technical key of the present invention is: no need for electroporation and special instruments and equipment, but the self-transformation characteristics of Moraxella catarrhalis are utilized to design a transformation method in which the carrier and the bacteria are directly mixed and incubated for 3-4 hours, and the transformation method can be completed through conventional screening. Compared with the existing technology, the experimental conditions of the present invention are low, the operation is simple, and the transformation success rate is high, and the transformation rate can reach more than 80%. In summary, the rapid and simple transformation method for Moraxella catarrhalis developed by the present invention, which is simple to operate and has high transformation efficiency, has important practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Flow chart of the rapid and convenient transformation method applied to Moraxella catarrhalis according to Example 1 of the present invention.

[0054] Figure 2 It is a structural diagram of the plasmid vector containing the mutant gene fragment of uspA2 constructed in Example 1 of the present invention.

[0055] Figure 3 It is a diagram of the electroporation results in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0057] The endpoints and any values ​​of the scope disclosed in this article are not limited to the precise scope or value, and these scopes or values ​​should be understood to include values ​​close to these scopes or values. For numerical ranges, between the endpoint values ​​of each scope, between the endpoint values ​​of each scope and a separate point value, and between separate point values, one or more new numerical ranges can be combined with each other, and these numerical ranges should be considered as specifically disclosed in this article. In the description of the application, unless otherwise stated, the meaning of similar words such as "multiple / multiple" is two / kinds or more than two / kinds.

[0058] The present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention. The experimental methods in the following examples where specific conditions are not specified are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0059] Example 1

[0060] A rapid and simple transformation method for Moraxella catarrhalis, the general steps are as follows Figure 1 As shown in the flowchart, the specific steps include:

[0061] (1) Design and synthesize the target mutant genes of uspA1 and uspA2 as templates for PCR amplification;

[0062] Among them, the target mutant gene of uspA1 is shown in SEQ ID No.1 in the sequence list, and the target mutant gene of uspA2 is shown in SEQ ID No.2 in the sequence list;

[0063] (2) designing and synthesizing upstream and downstream primers of the target mutant genes uspA1 and uspA2, introducing the upstream and downstream primers into the EcoRI / BamHI cloning site, and amplifying the target mutant genes uspA1 and uspA2 by PCR;

[0064] According to the target gene sequence, primers were designed using VectorNTI and their Tm values ​​and the possibility of dimer and hairpin formation were calculated. The primer specificity was tested using the blast function of NCBI. After confirmation, the primers were sent to Beijing Ruibo Company for primer synthesis.

[0065] The primer sequences are as follows:

[0066] 2105-EcoRI-F (SEQ ID No. 3 in the sequence listing)

[0067] gtggatcccccgggctgcaggaattcATGTCGAAGTTTCATCGCCAGGATG 2105-EcoRI-R (SEQ ID No. 4 in the sequence listing)

[0068] ctgtgggttgtgttctcgagTTAGTGTTTTGGTTCCACTTTCATG;

[0069] PCR amplification specifically includes:

[0070] 1) PCR amplification system (25 μl)

[0071] Reagents volume cDNA 1~4μL Primer-F(10uM) 0.5μL Primer-R(10uM) 0.5μL dNTP(2.5mM) 4μL 5×StimμLant 5μL 5×pfuBuffer 5μL Fastpfu 0.5μL <![CDATA[H2O]]> Make up the volume to 25 μL

[0072] 2) PCR conditions

[0073]

[0074] DNA electrophoresis

[0075] 1) According to the size of the DNA molecules to be separated, weigh an appropriate amount of agarose powder, put it into a conical flask, add an appropriate amount of 1×TAE electrophoresis buffer, and heat it in a microwave oven until it is completely dissolved and the solution is transparent. When the liquid cools to about 60°C, add EB substitute to the gel solution at a ratio of 1:20000 and shake well. Place the gel tank horizontally, insert the comb, and slowly pour the gel solution into the tank to form a uniform and horizontal gel surface. After the gel solidifies, carefully pull out the comb and put it into the electrophoresis tank.

[0076] 2) Add 1×TAE electrophoresis buffer into the tank until the liquid level covers the gel surface.

[0077] 3) Take an appropriate amount of DNA sample, mix it with 6× loading buffer and then load it.

[0078] 4) Connect the electrophoresis apparatus and electrophoresis tank, turn on the power supply, adjust the voltage output, and run the gel at 250V for 7 minutes.

[0079] Observe the movement of bromophenol blue. When it moves to about 4 cm from the front edge of the gel, end the electrophoresis.

[0080] 5) Place the DNA gel under 360nm UV light to observe the bands and take photos.

[0081] DNA gel purification

[0082] 1) Cut out the area where the target fragment is located from the DNA gel under ultraviolet light, put it into an EP tube, and weigh it.

[0083] 2) Add an equal volume of binding buffer according to the weight of the gel and place in a 65°C water bath until the gel is completely melted.

[0084] 3) Take out the melted gel and binding buffer mixture, wait for it to cool to room temperature, and add it to the recovery column.

[0085] 4) Centrifuge at 12,000 rpm for 1 min at room temperature and discard the liquid in the collection tube.

[0086] 5) Add 600 μl of washing buffer, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0087] 6) Repeat step 5) once.

[0088] 7) Replace the collection tube with a new 1.5 ml EP tube, add 50 μl of elution buffer to the column, and incubate at room temperature for 5 min.

[0089] 8) Centrifuge at 12,000 rpm for 2 min at room temperature. The DNA sample obtained in the EP tube can be used for subsequent experiments such as sequencing or enzyme digestion.

[0090] (3) After the uspA1 and uspA2 target mutant genes and the pUC57 plasmid vector were double-digested with EcoRI / BamHI, the target mutant genes were connected to the pUC57 plasmid vector to construct a plasmid vector carrying the uspA1 and uspA2 target mutant genes; specifically, the following contents were included:

[0091] Restriction enzyme digestion

[0092] 1) Check the required restriction enzyme concentration and applicable buffer on the NEB website as needed.

[0093] 2) Prepare 50 μL enzyme digestion system as needed:

[0094] Reagents volume DNA (fragment or vector) XμL(2μg) 10×Buffer 5μL 10×BSA 5μL Enzyme1 1μL Enzyme2 1μL <![CDATA[H2O]]> 38-XμL

[0095] 3) Digest in a 37°C water bath overnight.

[0096] The target fragment after enzyme digestion is recombined with the vector

[0097] 1) Recover the target fragment and vector after enzyme digestion by DNA gel extraction.

[0098] 2) The concentration of the obtained DNA solution was measured.

[0099] 3) Prepare 20 μl of ligation system according to the following conditions:

[0100] Reagents volume Purpose fragment XμL (molar ratio with carrier 3~7:1) Carrier YμL(100ng) 10×T4 ligase buffer 2μL T4 Ligase 1μL <![CDATA[H2O2]]> 17-X-YμL

[0101] Bacterial transformation

[0102] 1) Place competent cells of E. coli on ice and add 80 ng of plasmid.

[0103] 2) Incubate on ice for 30 minutes.

[0104] 3) Heat shock at 42°C for 45 seconds and immediately place on ice for 5 minutes.

[0105] 4) Add 500 μl LB medium and incubate at 37°C and 1000 rpm for 1 hour.

[0106] 5) Apply to LB Agar plates containing the corresponding resistance antibiotics.

[0107] 6) Grow inverted at 37°C overnight.

[0108] 7) After the clones are grown, use the tip of a gun to pick them up in LB medium with antibiotics and incubate at 37°C, 200 rpm

[0109] minutes, 12 hours until the bacterial solution becomes turbid.

[0110] Small plasmid

[0111] 1) Collect the bacterial solution, centrifuge at 6000 rpm for 5 minutes, and discard the supernatant.

[0112] 2) Add 250 μl of RNase-containing resuspension solution to resuspend the pellet and transfer the resuspension solution to a new EP tube.

[0113] 3) Add 250 μl of alkaline cell lysis buffer, invert 5 times, and let stand for 2 minutes until the liquid becomes transparent.

[0114] 4) Add 350 μl of neutralizing solution and invert 5 times to mix well.

[0115] 5) Centrifuge the precipitated suspension at 12,000 rpm for 10 minutes at room temperature.

[0116] 6) Add the equilibration solution to the DNA adsorption column, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant.

[0117] 7) Transfer the supernatant obtained in 5) to the DNA adsorption column equilibrated in 6) and centrifuge at 12,000 rpm for 1 minute at room temperature.

[0118] 8) Wash twice with 600 μl of cleaning solution and centrifuge at 12,000 rpm for 1 minute at room temperature.

[0119] 9) After discarding the waste liquid, spin at 10,000 rpm for 2 minutes to remove the cleaning solution.

[0120] 10) Open the tube cap, dry at room temperature for 5 minutes, replace the collection tube with a new 1.5 ml EP tube, add 50 μl elution buffer, centrifuge at 12,000 rpm for 1 minute at room temperature, and set aside the obtained plasmid vector. The constructed plasmid vector structure containing the mutant gene fragment of uspA2 is as follows: Figure 2 As shown, it contains sequence SEQ ID No.5.

[0121] (4) Pick a single colony of Moraxella catarrhalis that has been incubated overnight for 12 hours, inoculate it into a bacterial enrichment medium (brand name: OXOID; product number: PB0123A), the OD value of the Moraxella catarrhalis colony is 1.0, and place it in an air incubator at 37° C. for 18 hours;

[0122] (5) taking 10 μL of the constructed and extracted 1 μg / μL plasmid vector and dropping it in the central area of ​​the enrichment medium; the vector contains a resistance tag;

[0123] (6) Pick 5 colonies of Moraxella catarrhalis incubated for 16 hours on the enrichment medium and mix them with the plasmid vector carrying the target mutant genes of uspA1 and uspA2 until a biofilm with a diameter of 2 cm is formed; wherein the Moraxella catarrhalis is in the exponential growth phase, the OD value of the colony is 1.0, and the effective viable count is 1×10 8 cfu / ml;

[0124] (7) After the biofilm is dried at room temperature, the enrichment culture medium is placed in an air incubator at 37° C. and cultured for 3 hours;

[0125] (8) Then, growing colonies were visible to the naked eye, the OD value of the colonies was 1.0, and the number of effective viable bacteria was 1×10 8 cfu / ml;

[0126] Scrape off the colony and apply it on LB solid medium containing a resistance substance corresponding to the resistance tag;

[0127] In this embodiment, the preparation process of LB solid culture medium includes: mixing 100 ml of water and 4 g of LB solid culture medium powder, high pressure treatment, adding 100 μL of kanamycin after cooling, mixing evenly, then pouring 10 ml into a plate, shaking, and using after cooling.

[0128] (9) inverting the LB solid medium and placing it in an air incubator at 37° C. for overnight culture for 15 h;

[0129] Observe the colonies grown on the LB solid medium plate, pick a single colony for passage, and then sequence and verify;

[0130] The colonies were counted and the transformation efficiency was calculated. Transformation rate = number of successful transformations / total number of transformations. After calculation, the transformation rate in this example was 82.6%.

[0131] (10) Design primers for the target mutant gene for PCR amplification and Sanger sequencing to verify whether the knockout is successful on the chromosome of Moraxella catarrhalis; if the sequencing result is consistent with the reference sequence, it means that the knockout is successful. It has been verified that the target mutant genes uspA1 and uspA2 in this example were successfully knocked out on the chromosome of Moraxella catarrhalis.

[0132] Comparative Example 1

[0133] Preparation of competent cells of Moraxella catarrhalis and routine electroporation experiments

[0134] 1. Prepare LB liquid culture medium first. Add 25g of broth culture medium powder to 1L of mineral water, stir evenly, and set aside after high pressure at 120℃ for 30min. At the same time, prepare LB solid culture medium in advance. Add 4g of solid culture medium powder to 100mL of mineral water, stir evenly, and cool slightly after high pressure at 120℃ for 30min, add antibiotics and stir evenly, pour into culture dishes, pour 10mL of culture medium into each culture dish, and then shake to cover the entire culture dish;

[0135] 2. Pick an inoculation loop of Moraxella catarrhalis and make three-zone lines in the above-mentioned culture medium so that the bacterial liquid covers the entire culture dish for bacterial enrichment and place in a 37°C incubator for overnight culture. The next day, pick a single colony in the culture medium and place it in 600 μL of LB broth at 37°C and shake it overnight at 1000 rpm;

[0136] 3. Transfer 600 μL of the overnight shaken strain of Catarrhalis into 60 mL of LB broth and shake it on a shaker at 37°C and 1000 rpm for 3 hours. Measure the absorbance value on a spectrophotometer. When the broth OD 600 reaches 0.6, immediately put the broth on ice for 30 minutes and centrifuge it at 5000 rpm and 4°C for 10 minutes in a small desktop centrifuge. After the centrifugation, remove the supernatant.

[0137] 4. Use a dropper to resuspend the precipitate in 10 mL of pre-cooled sterile 10% glycerol. Repeat the centrifugation and resuspension three times (centrifugation resuspension-centrifugation resuspension-centrifugation). Be gentle during the glycerol resuspension and centrifugation transfer process.

[0138] 5. Finally, resuspend the precipitate with 600 μL of pre-cooled 10% sterile glycerol, take 50 μL of electroporated competent cells and aliquot them, and store them at -80°C;

[0139] 6. Thaw 50 μL of electroporated competent cells on ice for a few minutes before electroporation. After thawing, mix the competent cells with the plasmid, with the volume ratio of competent cells to plasmid being 10:1 (the plasmid DNA content should not exceed 100 ng). After mixing, gently mix well and let stand on ice for 5 minutes;

[0140] 7. Transfer the mixed competent cells and plasmid into a 2mm electroporation cup and set the electroporator to 2.5kv, 2002, 25uF for electroporation;

[0141] 8. After electroporation, transfer the cells into 1 mL of LB broth and shake on a shaker at 37°C for 2.5 hours.

[0142] 9. Take 100 μL of the mixture in the above LB broth medium and place it on LB solid medium with antibiotics, streak and mix well, and perform drug screening. If colonies grow, it is determined to be Moraxella catarrhalis that has been successfully electroporated.

[0143] After preparing Moraxella catarrhalis competent cells by electroporation, plasmid transformation was performed and verified on screening plates. It was found that the transformation efficiency was low (the electroporation transformation rate was only 16.7%). Almost no colonies grew on the four screening plates, and only one screening plate grew about 10 colonies. Figure 3 shown.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A rapid and simple transformation method for Moraxella catarrhalis, characterized in that: The following steps are involved: The vector carrying the target gene or the ligation system containing the vector carrying the target gene is mixed evenly with the colony of Moraxella catarrhalis, and then incubated at 32-39° C. for 2-5 hours; The catarrhalis bacteria were in the exponential growth phase, the OD value of the colony was between 0.8 and 1.2, and the effective viable count was 0.5×10 8 cfu / ml-3×10 8 cfu / ml.

2. The rapid and simple transformation method for Moraxella catarrhalis according to claim 1, characterized in that: The target gene includes at least one of the mutant gene fragments of uspA1 and uspA2.

3. The rapid and simple transformation method for Moraxella catarrhalis according to claim 1, characterized in that: The vector includes a pUC plasmid vector.

4. The rapid and convenient transformation method for Moraxella catarrhalis according to claim 1, characterized in that: The vector carrying the target gene or the ligation system containing the vector carrying the target gene is evenly mixed with the colony of Moraxella catarrhalis, and then incubated at 37-38° C. for 3-4 hours.

5. The rapid and convenient transformation method for Moraxella catarrhalis according to claim 1, characterized in that: The following steps are involved: (1) Design and synthesize the target mutant gene as an amplification template; (2) amplifying the target mutant gene; (3) connecting the target mutant gene to a vector to construct the vector carrying the target mutant gene; (4) Pick a single colony of Moraxella catarrhalis that has been incubated overnight for 12-24 hours, inoculate it into a bacterial enrichment medium, wherein the OD value of the Moraxella catarrhalis colony is between 0.8 and 1.2, and place it in an air incubator at 35-39° C. for 10-30 hours; (5) taking 5-25 μL of the constructed and extracted 0.8-2 μg / μL vector and dropping it in the central area of ​​the enrichment medium; the vector contains a selection tag; (6) Pick 2-15 colonies of Moraxella catarrhalis on the enrichment medium and mix them evenly with the vector carrying the target mutant gene until a biofilm with a diameter of 1-3 cm is formed; wherein the Moraxella catarrhalis is in the exponential growth phase, the OD value of the colony is between 0.8 and 1.2, and the effective viable count is 0.5×10 8 cfu / ml-3×10 8 cfu / ml; (7) After the biofilm is dried at room temperature, the enrichment culture medium is placed in an air incubator at 32-39° C. for 2-5 hours; (8) Then, growing colonies were observed, the OD value of which was between 0.8 and 1.2, and the effective viable count was 0.5×10 8 cfu / ml-3×10 8 cfu / ml; Scrape off any colonies and spread them on medium containing the screening substance corresponding to the screening label; (9) Place the culture medium containing the screening substance corresponding to the screening tag described in step (8) in an air incubator at 32-39°C for overnight culture for 12-24 hours.

6. The rapid and convenient transformation method for Moraxella catarrhalis according to claim 5, characterized in that: In step (3), it also includes screening and sequencing to identify and construct the vector carrying the target mutant gene.

7. The rapid and convenient transformation method for Moraxella catarrhalis according to claim 5, characterized in that: In step (9), the following steps are also included: Observe the colonies grown on the culture medium containing the screening substance corresponding to the screening tag, pick a single colony for passaging, and then sequence and verify; The colonies were counted and the transformation efficiency was calculated.

8. The rapid and convenient transformation method for Moraxella catarrhalis according to claim 5, characterized in that: The following steps are also included: For the target mutant gene, primers are designed for amplification and sequencing to verify whether the knockout is successful on the chromosome of Moraxella catarrhalis.

9. The rapid and convenient transformation method for Moraxella catarrhalis according to claims 5-8, characterized in that: The following steps are involved: (1) Design and synthesize the target mutant genes of uspA1 and uspA2 as templates for PCR amplification; (2) Designing and synthesizing upstream and downstream primers of the target mutant genes uspA1 and uspA2, introducing the upstream and downstream primers into the EcoRI / BamHI cloning site, and amplifying the target mutant genes uspA1 and uspA2 by PCR; (3) After double digesting the uspA1 and uspA2 target mutant genes and the pUC57 plasmid vector with EcoRI / BamHI, the target mutant genes were connected to the pUC57 plasmid vector to construct a plasmid vector carrying the uspA1 and uspA2 target mutant genes; screening and sequencing to identify the constructed plasmid vector carrying the uspA1 and uspA2 target mutant genes; (4) Pick a single colony of Moraxella catarrhalis that has been incubated overnight for 12 hours, inoculate it into a bacterial enrichment medium, and place the colony of Moraxella catarrhalis with an OD value between 0.8 and 1.0 in an air incubator at 37°C for 16 to 20 hours; (5) Take 10-20 μL of the constructed and extracted 1 μg / μL plasmid vector and drop it in the central area of ​​the bacterial enrichment medium; the vector contains a resistance tag; (6) On the enrichment medium, 5-10 Moraxella catarrhalis colonies incubated for 16-20 hours are picked and mixed with the plasmid vector carrying the target mutant genes of uspA1 and uspA2 until a biofilm with a diameter of 2 cm is formed; wherein the Moraxella catarrhalis is in the exponential growth phase, the OD value of the colony is between 0.8 and 1.0, and the effective viable count is 0.5×10 8 cfu / ml-1.5×10 8 cfu / ml; (7) After the biofilm is dried at room temperature, the enrichment culture medium is placed in an air incubator at 37° C. for 3-4 hours; (8) Then, growing colonies were visible to the naked eye, the OD value of the colonies was between 0.8 and 1.0, and the effective viable count was 0.5×10 8 cfu / ml-1.5×10 8 cfu / ml; Scrape off the colony and apply it on LB solid medium containing a resistance substance corresponding to the resistance tag; (9) Invert the LB solid medium and place it in an air incubator at 37° C. for overnight culture for 15 h; Observe the colonies grown on the LB solid medium plate, pick a single colony for passage, and then sequence and verify; The colonies were counted and the transformation efficiency was calculated; (10) Design primers for PCR amplification and Sanger sequencing of the target mutant gene to verify whether the knockout is successful on the chromosome of Moraxella catarrhalis; if the sequencing result is consistent with the reference sequence, it means that the knockout is successful.

10. A genetically transformed strain of Moraxella catarrhalis, characterized in that: The method is constructed by the rapid and convenient transformation method applied to Moraxella catarrhalis according to any one of claims 1 to 9.

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