CN111621510B - Optimized clostridium Yankeei exogenous DNA electrotransformation method - Google Patents

Abstract

The invention provides an application of a clostridium Yankeei self-restriction modification system, which is used for modifying exogenous DNA of clostridium Yankeei so as to improve the electric conversion efficiency of the exogenous DNA to clostridium Yankeei. The invention also provides a method for improving the electric conversion efficiency of the exogenous DNA of clostridium Yankee, which comprises the following steps: (a) Providing an exogenous DNA modified by a clostridium Yankeei self-restriction modification system; (b) Electrotransformation of the exogenous DNA provided in step (a) into clostridium Yankee; (c) cell resuscitation of said clostridium Yankeei. The method of the invention can obviously improve the electric conversion efficiency of exogenous DNA to clostridium Yankee, which is improved by 2 orders of magnitude compared with the prior art.

Description

An optimized method for electrotransformation of exogenous DNA of Clostridium ljungdahlii

Technical field

The invention belongs to the field of biotechnology, and specifically relates to an optimized electrotransformation method of exogenous DNA of Clostridium jungdahlii (Clostridium ljungdahlii).

Background technique

In recent years, the extensive use of fossil fuels has caused serious environmental problems, such as air pollution, greenhouse effect, etc.; at the same time, the non-renewable nature of fossil fuels has also caused people to face energy shortages in the foreseeable future. Therefore, the search for renewable and clean energy is urgent. Obviously, if the waste gas rich in one-carbon gases such as CO ₂ and CO can be converted into energy or chemical raw materials that can be utilized by us, this series of problems can be perfectly solved. Fortunately, the fermentation process of Clostridium aerogenes is exactly in line with this point. They can produce various organic compounds while consuming one-carbon gas, many of which are high-value products that can be used as fuel and chemical raw materials.

Clostridium ljungdahlii is a member of Clostridium aerogenes. It is an industrial bacterium with great potential and competitiveness. It can use one-carbon gas (CO, CO ₂ ) for anaerobic fermentation through special Wood-Ljungdahl solids. The carbon pathway generates the central metabolite acetyl-CoA, which ultimately synthesizes biofuels and chemical raw materials such as ethanol and acetic acid. Compared with other Clostridium aerogenes, the molecular operating system of Clostridium ljungdahlii is relatively complete, and people can now perform basic operations such as introducing foreign plasmids and simple gene editing.

However, the efficiency of existing transformation methods for C. ljungdahlii is low. Take the most commonly used E. coli-Clostridium shuttle plasmid PMTL83151 plasmid as an example: the latest efficiency reported by Bastian Molitor et al. in 2016 is only 3.23±2.02×10 ² CFU /μg; the highest efficiency reported so far is 3.1±1.8×10 ³ CFU/μg achieved by Ching Leang et al. in 2013, which was mainly achieved by increasing the concentration of competent bacteria, with a concentration factor of 1000 times about. This greatly limits the conduct of related research, such as the establishment of a mutant library with effective library capacity based on Clostridium ljungdahlii and the editing of large genome fragments that require high transformation efficiency. Therefore, establishing an efficient transformation method for C. ljungdahlii has become a bottleneck that urgently needs to be solved.

Therefore, there is an urgent need in this field to develop an efficient and easy-to-operate electrotransformation method for exogenous DNA of C. ljungdahlii.

Contents of the invention

The purpose of the present invention is to provide an efficient and easy-to-operate electrotransformation method of exogenous DNA of Clostridium ljungdahlii.

In a first aspect of the present invention, there is provided the use of a Clostridium ljungdahlii self-restriction modification system for modifying exogenous DNA of Clostridium ljungdahlii to improve the ability of the exogenous DNA to electrical conversion efficiency.

In another preferred embodiment, the Clostridium ljungdahlii includes: Clostridium ljungdahlii ATCC 55383, Clostridium ljungdahlii ATCC 49587, and Clostridium ljungdahlii DSM 13528.

In another preferred embodiment, the modification is methylation modification.

In another preferred embodiment, the Clostridium ljungdahlii self-restriction modification system is a type II Clostridium ljungdahlii self-restriction modification system.

In another preferred example, the Clostridium ljungdahlii self-restriction modification system includes methylase encoding genes CLJU_c08460 and CLJU_c24700.

In another preferred example, the Clostridium ljungdahlii self-restriction modification system is the methylase encoding gene CLJU_c24700.

In another preferred embodiment, the exogenous DNA is a shuttle plasmid.

In another preferred example, the exogenous DNA is derived from: plasmid pMTL83151 (http://clostron.com/pMTL80000.php).

In another preferred embodiment, the exogenous DNA includes plasmid pMTL83151 and DNA sequences from other sources that are subsequently integrated into pMTL83151.

In another preferred embodiment, the modification is completed in a modified plasmid host, and the modified plasmid host includes: Escherichia coli.

In another preferred example, the modified plasmid host is E. coli JM110 (genotype dam ^- , dcm ^- ).

In another preferred example, the electroconversion efficiency can be increased to ≥10 ⁴ CFU/μg, preferably ≥1.73×10 ⁴ CFU/μg, and more preferably ≥2.5×10 ⁴ CFU/μg.

In a second aspect of the present invention, a method for improving the electrotransformation efficiency of exogenous DNA of Clostridium ljungdahlii is provided, including the steps:

(a) providing an exogenous DNA modified by Clostridium ljungdahlii's own restriction modification system;

(b) electrotransforming the exogenous DNA provided in step (a) into Clostridium ljungdahlii;

(c) Cell recovery of C. ljungdahlii.

In another preferred example, in the step (a), the Clostridium ljungdahlii includes: Clostridium ljungdahlii ATCC 55383, Clostridium ljungdahlii ATCC 49587, and Clostridium ljungdahlii DSM 13528.

In another preferred embodiment, in step (a), the modification is methylation modification.

In another preferred embodiment, in step (a), the Clostridium ljungdahlii self-limiting modification system is a type II Clostridium ljungdahlii self-restricting modification system.

In another preferred example, in step (a), the Clostridium ljungdahlii self-restriction modification system includes methylase encoding genes CLJU_c08460 and CLJU_c24700.

In another preferred example, in step (a), the Clostridium ljungdahlii self-restriction modification system is the methylase CLJU_c24700 gene.

In another preferred embodiment, in step (a), the exogenous DNA is a shuttle plasmid.

In another preferred example, in step (a), the exogenous DNA is derived from: plasmid pMTL83151 (http://clostron.com/pMTL80000.php).

In another preferred embodiment, in step (a), the exogenous DNA includes plasmid pMTL83151 and DNA sequences from other sources that are subsequently integrated into pMTL83151.

In another preferred embodiment, in step (a), the modification is completed in a modified plasmid host, and the modified plasmid host is Escherichia coli.

In another preferred example, in step (a), the modified plasmid host is E. coli JM110 (genotype dam ^- , dcm ^- ).

In another preferred embodiment, the step (b) includes the step of: (b1) adding a weakening agent to the Clostridium ljungdahlii cells to weaken the cell wall of the C. ljungdahlii.

In another preferred embodiment, the weakening treatment is performed when the bacterial cells grow to an OD600 of 0.35-0.55, preferably when the bacterial cells grow to an OD600 of 0.4-0.5.

In another preferred example, the weakening treatment time is 60-120 min, preferably 80-100 min, more preferably 90 min.

In another preferred example, the temperature of the weakening treatment is 32-45°C, preferably 35-40°C, and more preferably 37°C.

In another preferred embodiment, the weakening agent includes glycine, DL-threonine, β-lactam antibiotics, lysozyme, and isonicotinic acid.

In another preferred embodiment, the weakening agent is glycine, and the final concentration is 0.5-5% w/v, preferably 0.8-2.5% w/v, more preferably 1-1.5% w/v, more preferably Optimum is 1.25% w/v.

In another preferred embodiment, step (b1) also includes adding a buffer protective agent.

In another preferred embodiment, the buffer protective agent is sucrose.

In another preferred embodiment, the buffer protective agent is sucrose, and the final concentration is 0.1-0.5M, preferably 0.2-0.4M, more preferably 0.3M.

In another preferred embodiment, in step (b), in the electrical conversion, the electric shock voltage is 500-1500V, preferably 800-1200V, and more preferably 1000V.

In another preferred example, in step (b), in the electrical conversion, the electric shock capacitance is 30-100 μF, preferably 40-60 μF, and more preferably 50 μF.

In another preferred example, in step (b), in the electrical conversion, the electric shock resistance is 120-300Ω, preferably 150-250Ω, and more preferably 200Ω.

In another preferred embodiment, in step (c), the cell recovery time is 15-25h, preferably 18-22h, and more preferably 20h.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of drawings

Figure 1 shows the effect of cell wall weakening of C.ljungdahlii DSM 13528 on the electroconversion efficiency of foreign DNA. Among them, after being treated with 1.25% v/v glycine and 0.3M sucrose for 90 minutes, the electroconversion efficiency reached 1.93×10 ³ CFU/μg.

Figure 2 shows the comparison between the conversion efficiency obtained using the optimized electroporation parameters provided by the present invention and the conversion efficiency obtained using the existing more efficient electroporation parameters.

Figure 3 shows the effect of recovery time on transformation efficiency of C.ljungdahlii DSM 13528 after electrotransformation. Among them, the left pictures a, b, c, and d are the transformants obtained after 100 μl of the recovery system was plated after 6h, 12h, 20h, and 30h recovery respectively; the right picture is the statistical result of the transformation rate.

Figure 4 shows the methylation process using C.ljungdahlii DSM 13528 methylase to methylate exogenous DNA for electroconversion.

Figure 5 shows the conversion rates after electroconversion using C.ljungdahlii DSM 13528 methylase to methylate exogenous DNA.

Detailed ways

After extensive and in-depth research and extensive screening, the inventor developed for the first time an efficient and easy-to-operate electroconversion method for exogenous DNA of Clostridium ljungdahlii. Experiments show that using the C. ljungdahlii self-restriction modification system selected in the present invention to perform methylation modification on exogenous DNA can effectively improve the efficiency of electrotransformation of exogenous DNA in C. ljungdahlii. In addition, experiments have shown that adding 1.25% v/v glycine and 0.3M sucrose to the Youngella bacteria to weaken the cell wall before the electrotransformation step can further improve the transformation efficiency of exogenous DNA; and, during electroporation In the transformation step, when the electric shock voltage is 1000V, the electric shock capacitance is 50 μF, and the electric shock resistance is 200Ω, the conversion efficiency of exogenous DNA is the highest. On this basis, the present invention was completed.

the term

As used herein, the terms "Clostridium ljungdahlii" and "Clostridium ljungdahlii" are used interchangeably and refer to the strain to be transformed with its own limiting modification system as described in the present invention.

self-limiting modification system

As used herein, the terms "methylase" and "methyltransferase" are used interchangeably and refer to the self-restriction modification system in Clostridium ljungdahlii described in the present invention that can methylate DNA. modified enzyme.

For bacteria of the genus Clostridium, an important factor limiting their transformation efficiency is their own powerful restriction modification system. The restriction modification system can recognize heterologous DNA molecules, and then cut the invading DNA under the action of corresponding restriction enzymes, ultimately ensuring the stability of its own genetic system.

In the present invention, based on this principle, a method for protecting exogenous DNA by simulating host methylation modification is provided.

In Clostridium ljungdahlii, there are seven restriction modification systems, including one type I (CLJU_c03310~CLJU-c03330) and six type II (CLJU_c03480, CLJU_c08460, CLJU_c24700, CLJU_c30980, CLJU_c32690, CLJU_c33980). See Table 3 for details. .

In the present invention, M4 (CLJU_c24700) methylase, which can significantly improve the transformation efficiency of the test plasmid, was screened out from seven restriction modification systems of Clostridium ljungdahlii, followed by M3 (CLJU_c08460).

Method of the present invention

The invention provides a method for improving the electrotransformation efficiency of exogenous DNA of Clostridium ljungdahlii, which includes the steps:

(a) providing an exogenous DNA modified by Clostridium ljungdahlii's own restriction modification system;

(b) electrotransforming the exogenous DNA provided in step (a) into Clostridium ljungdahlii;

(c) Cell recovery of C. ljungdahlii.

In one embodiment of the invention, the modification is methylation modification.

In one embodiment of the present invention, the Clostridium ljungdahlii self-restriction modification system is a type II Clostridium ljungdahlii self-restriction modification system.

In a preferred embodiment of the present invention, the Clostridium ljungdahlii self-restriction modification system includes methylase encoding genes CLJU_c08460 and CLJU_c24700. In a more preferred embodiment, the Clostridium ljungdahlii self-restriction modification system is the methylase CLJU_c24700 gene.

In another preferred example, the exogenous DNA is derived from: plasmid pMTL83151.

In another preferred embodiment, the exogenous DNA includes plasmid pMTL83151 and DNA sequences from other sources that are subsequently integrated into pMTL83151.

In another preferred embodiment, the modification is completed in a modified plasmid host, and the modified plasmid host is Escherichia coli. In another preferred example, the modified plasmid host is E. coli JM110 (genotype dam ^- , dcm ^- ).

In addition, Clostridium ljungdahlii is a member of the Firmicutes phylum. In addition to the self-limiting modification system described above, the thicker cell wall outside its cell membrane prevents foreign DNA from entering the cell body, which is another factor that limits its transformation efficiency. An important reason.

Therefore, in order to improve the transformation efficiency of C. ljungdahlii, it is necessary to weaken its cell wall; currently commonly used cell wall weakening agents include glycine, DL-threonine, penicillin, lysozyme, etc. It is worth mentioning that these cell wall weakening agents are often accompanied by cytotoxicity, and improper operation can easily cause cell lysis. Therefore, it is often necessary to consider factors such as adding buffer protective agents, controlling processing time, and reagent concentration during processing. In the present invention, after extensive screening, the inventor finally selected glycine as the cell wall weakening agent and added sucrose as the buffering and protecting agent. Among them, glycine can reduce the cross-linking of peptidoglycan in the cell wall, reduce the strength of the cell wall, and ultimately improve the electroshock conversion rate.

In one embodiment of the present invention, the method of the present invention includes the step of: (b1) adding a weakening agent to the Clostridium ljungdahlii cells to weaken the cell wall of the C. ljungdahlii.

In one embodiment of the present invention, the weakening treatment is performed when the bacterial cells grow to an OD600 of 0.35-0.55, preferably when the bacterial cells grow to an OD600 of 0.4-0.5; the time of the weakening treatment It is 60-120min, preferably 80-100min, more preferably 90min; the temperature of the weakening treatment is 32-45°C, preferably 35-40°C, more preferably 37°C.

In another preferred embodiment, the weakening agent includes glycine, DL-threonine, β-lactam antibiotics, lysozyme, and isonicotinic acid. Preferably it is glycine.

In another preferred embodiment, the weakening agent is glycine, and the final concentration is 0.5-5% w/v, preferably 0.8-2.5% w/v, more preferably 1-1.5% w/v, more preferably Optimum is 1.25% w/v.

In a preferred embodiment, step (b1) also includes adding a buffer protective agent; in another preferred example, the buffer protective agent is sucrose, and its final concentration is 0.1-0.5M, preferably 0.2-0.4M, preferably 0.3M.

In addition, electroporation conditions are an important factor affecting transformation efficiency. It is related to whether the plasmid can successfully enter the cells and whether subsequent cell recovery can proceed smoothly. Electroporation conditions usually involve the following aspects: shock voltage, shock capacitance, shock resistance, recovery time, etc.

In one embodiment of the present invention, in the electroconversion, the electric shock voltage is 500-1500V, preferably 800-1200V, more preferably 1000V; the electric shock capacitance is 30-100 μF, preferably 40-60 μF , preferably 50μF. ; The electric shock resistance is 120-300Ω, preferably 150-250Ω, and more preferably 200Ω.

In addition to the above electroporation parameters, cells need a period of time to recover the broken cell wall and express plasmid resistance after electroporation transformation. This period of time is called recovery time. The length of recovery time will also affect the final conversion efficiency. In order to explore the impact of recovery time on conversion efficiency, the inventors established a series of recovery time gradients. Finally, it was found that after appropriately extending the recovery time, the electroporation transformation efficiency of C. ljungdahlii can be greatly improved.

In one embodiment of the present invention, the cell recovery time is 15-25h, preferably 18-22h, and more preferably 20h.

The main advantages of the present invention include:

1) Utilize the self-restriction modification system of C. ljungdahlii to modify exogenous DNA to improve the conversion efficiency of exogenous DNA into C. ljungdahlii; and, in the multiple self-restriction modification systems of C. ljungdahlii , for the first time, a self-restriction modification system was screened that can significantly improve the conversion efficiency of foreign DNA compared with other self-restriction modification systems.

2) Optimized the concentration of weakening agent and buffer protective agent that are more effective in weakening the cell wall of Clostridium ljungdahlii.

3) Optimize relatively efficient electrical conversion conditions, which involve shock voltage, shock capacitance, shock resistance, and recovery time.

4) Using the method of the present invention, the electrotransformation efficiency of exogenous DNA into Clostridium ljungdahlii can be increased to 1.73±0.04×10 ⁴ CFU/μg, which is an improvement of 2 orders of magnitude compared with the existing technology.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. Suggested conditions. Unless otherwise stated, percentages and parts are by weight.

Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

Materials and Methods

Material

1. Strains and plasmids

Table 1 Bacterial strains and plasmids

2. Primers

The primers used in the experiment are shown in the table below. All primers were synthesized by Shanghai Huajin Biotechnology Company.

Table 2 Primers used in the present invention

3. Reagents and equipment

The gel recovery kit and plasmid extraction kit used in the experiment are products of Axygen; the restriction enzymes are products of Thermo Fisher Scientific; the KOD FX and KOD-plus-neo used in the PCR reaction are products of TOYOBO; The Taq DNA polymerase used in the E. coli colony PCR reaction is a product of TRANSGEN BIOTECH; the Solution I used in the ligation reaction is a product of Takara; YTF medium (Yeast extract 10g/L, Tryptone16g/L, Fructose 10g/L, NaCl 0.2g/ L, L-cysteine 0.3g/L, trace element storage solution 2ml/L, vitamin storage solution 1ml/L, adjust pH to 6.0 with 6M HCl); trace element storage solution (nitrilotriacetic acid 10.00g/L, MnSO ₄ 5.00g/L, (NH ₄ ) ₂ SO ₄ ·FeSO ₄ ·6H ₂ O 4.00g/L, CoCl ₂ ·6H ₂ O 1.00g/L, ZnSO ₄ ·7H ₂ O 1.00g/L, CuCl ₂ ·2H ₂ O 0.10g/L, NiCl ₂ ·6H ₂ O 0.10g/L, Na ₂ MoO ₄ ·2H ₂ O 0.10g/L, Na ₂ O ₄ Se 0.10g/L, Na ₂ WO ₄ ·2H ₂ O 0.10g/L, pH 6.0. Filter and sterilize. When preparing the trace element storage solution, mix and dissolve other ingredients except nitrilotriacetic acid to a constant volume, then adjust the pH to 6.0, and quickly add nitrilotriacetic acid); vitamin storage Liquid (VB6 100mg/L, thiamine 50.00mg/L, VB2 50.00mg/L, calcium pantothenate 50.00mg/L, lipoic acid 50.00mg/L, para-aminobenzoic acid 50.00mg/L, niacin 50.00mg/ L, VB12 50.00mg/L, biotin 20.00mg/L, folic acid 20.00mg/L. After preparation, filter and sterilize with a 0.22μm filter before use); SMP buffer (Na ₂ HPO ₄ 1mM, NaH ₂ PO ₄ 1mM, MgCl ₂ 1mM, Sucrose 270mM); electroshocker Gene PulserTM, electroshock cup (0.2cm), BIO-RAD PCR instrument, special gel scanner GS710scanner and PDquest software BioRad products; room temperature and refrigerated centrifuges are products of Eppendorf; anaerobic The incubator is the Whitley A35anaerobic workstation product of GE Company, and the spectrophotometer is U-1800spectrophotometer of HITACHI Company.

method

1. Electroporation competent preparation

Inoculate a single colony of Clostridium ljungdahlii into 5 ml of YTF liquid culture medium, and culture it anaerobically at 37°C overnight to obtain a seed liquid; inoculate it into 25 ml YTF culture medium (50 ml centrifuge tube) according to an inoculation amount of 1‰, and culture it anaerobically at 37°C. . When OD ₆₀₀ reaches 0.4-0.5, transfer the cells to ice and cool for 10 minutes; then centrifuge to collect the cells. The specific parameters are centrifugation at 5000 rpm for 6 minutes. Use SMP buffer (pre-cooled on ice) to wash the cells twice, and finally use 450 μl of SMP buffer plus 50 μl of DMSO to resuspend the cells (pre-cooled on ice), put them in sterile storage tubes, and store at -80°C.

2.Electroconversion method

Mix 200 μl of competent cells melted on ice with the test plasmid PMTL83151. The total amount of plasmid is 1 μg and the volume does not exceed 10 μl. The mixed system is allowed to stand on ice for 5 minutes, then added to an electroporation cup (pre-cooled on ice) and electroporated. . The electroconversion parameters are voltage 1000v, capacitance 50μF, resistance 200Ω, and electrode spacing 2mm. After electroporation, quickly mix the electroporation system with 1 ml of YTF liquid culture medium and resuscitate it in a 5 ml Eppendorf tube. After an appropriate period of anaerobic resuscitation at 37°C, take 100 μl of the recovery system and apply it on a YTF solid plate. The concentration of methylsulfonate and chloramphenicol on the plate is 5 μg/ml. The conversion rate was calculated after 5 days of anaerobic cultivation at 37°C.

3. Artificially modified plasmid construction

We extracted the genome of C.ljungdahlii as a template and used KOD neo enzyme to perform a PCR reaction to obtain the potential Mtase gene fragment. Then, the obtained target fragment was digested with SalⅠ and SmaⅠ and cleaned and recovered. At the same time, SalⅠ and SmaⅠ were used to linearize pMV118. vector, and finally use ligase Solution I to ligate the fragment to the vector overnight at 16°C. The ligation product is transformed into Escherichia coli Top10 competent cells and spread on LB medium containing 100 μg/ml ampicillin. The single colony grown is then verified by colony PCR. , extract the plasmid and obtain the correct artificially modified plasmid after sequencing and verification.

Example 1: Effect of glycine as a cell wall weakening agent on electroconversion efficiency

In this example, Clostridium ljungdahlii cells are first grown in YTF liquid culture medium. When the cells grow to an OD ₆₀₀ of 0.4-0.5, glycine and sucrose stock solutions are added to the final concentrations of 1.25% and 0.3M respectively. Treat at 37°C for 90 minutes (cells basically stop growing during this process), and then collect the bacteria to make competent cells. Test the competent efficiency. The experimental results are shown in Figure 1.

As shown in Figure 1, after being treated with 1.25% glycine and 0.3M sucrose for 90 minutes, the electroconversion efficiency reached 1.93×10 ³ CFU/μg.

Experimental results show that using glycine as a cell wall weakening agent can significantly improve the transformation efficiency of Clostridium ljungdahlii; the transformation efficiency at this time is in the same order of magnitude as the highest efficiency reported by Ching Leang et al. However, the competent bacteria used in our experiments The body concentration is about 1/100 of what has been reported, which greatly simplifies the experimental process while ensuring efficiency.

Example 2: Effect of electroconversion conditions on electroconversion efficiency

In this embodiment, the parameters of shock voltage, shock capacitance, and shock resistance in electroporation conditions are optimized.

First, the inventors tried the electroconversion voltage, resistance, capacitance and other related parameters that have been reported to achieve good conversion effects. In the existing reports, the plasmid electroconversion efficiency when infinite resistance, 1.8kv voltage, and 25μF capacitance were used Highest. At the same time, the optimized parameters provided by the present invention (voltage 1000v, capacitance 50μF, resistance 200Ω) were used to conduct electrical conversion experiments, and the conversion efficiencies of the two were compared.

The experimental results are shown in Figure 2. The results show that compared with the existing more efficient parameters and the optimal parameters provided by the present invention, not only do they have no significant advantages but they are even worse.

Example 3: Effect of appropriately extending recovery time on electroconversion efficiency

In this example, a series of recovery time gradients were established to explore the impact of recovery time on conversion efficiency.

Finally, it was found that after appropriately extending the recovery time, the electroporation transformation efficiency of C. ljungdahlii can be greatly improved. The experimental results are shown in Figure 3.

Figure 3 shows the effect of recovery time on transformation efficiency of C.ljungdahlii DSM 13528 after electrotransformation. Among them, the left pictures a, b, c, and d are the transformants obtained after 100 μl of the recovery system was plated after 6h, 12h, 20h, and 30h recovery respectively; the right picture is the statistical result of the conversion rate.

It can be seen that the recovery time has a great impact on the electric shock transformation efficiency of C. ljungdahlii. When the recovery time is extended to 20 hours, the transformation efficiency is improved by an order of magnitude compared to the previous one, reaching the level of 10 ⁴ .

Example 4: Heterologous simulation of Clostridium ljungdahlii self-restriction modification system to protect foreign DNA

In this example, the inventor first used bioinformatics methods (http://rebase.neb.com/rebase/rebase.html) to screen possible restriction modification systems in Clostridium ljungdahlii (the results are shown in Table 3 ).

There are seven restriction modification systems in Clostridium ljungdahlii, including one type I (CLJU_c03310) and six type II (CLJU_c03480, CLJU_c08460, CLJU_c24700, CLJU_c30980, CLJU_c32690, CLJU_c33980).

Table 3 MTases in the C.ljungdahlii DSM 13528 genome

MTase serial number type Modification basics recognition site M1 CLJU_c03310 Ⅰ m6A M2 CLJU_c03480 Ⅱ m4C/m6A M3 CLJU_c08460 Ⅱ m6A M4 CLJU_c24700 Ⅱ m6A GTTAAT M5 CLJU_c30980 Ⅱ m4C/m6A M6 CLJU_c32690 Ⅱ m4C/m6A M7 CLJU_c33980 Ⅱ m4C/m6A

Then, specific strains and plasmids were selected to construct the C. ljungdahlii artificial simulation modification system.

In this example, E. coli JM110 (genotype: dam ^- , dcm ^- ) was selected as the modified plasmid host; next, the low-copy, ampicillin-resistant pMV118 plasmid was selected as the artificial modified plasmid starting vector ( This is to distinguish it from the chloramphenicol-resistant high-copy test plasmid pMTL83151). After using lacZ to detect the availability of its lactose-inducible promoter (experimental data not shown), the effects of different methyltransferases on the electrotransformation efficiency of C. ljungdahlii were tested according to the experimental process in Figure 4. The specific experimental results are shown in Figure 5.

The results show that among the seven methyltransferases, M4 (CLJU_c24700) can significantly improve the transformation efficiency of the test plasmid, increasing the electrotransformation efficiency of Clostridium ljungdahlii to 1.73±0.04×10 ⁴ CFU/μg, meeting the experimental needs .

discuss

The embodiments of the present invention provide a complete set of methods for efficient electrotransformation of Clostridium ljungdahlii based on the pMTL83151 plasmid. In the present invention, glycine is used as a cell wall weakening agent, the electroporation conditions optimized by the invention are selected, the recovery time is extended to 20 hours, and its own methylation system is used to pretreat the plasmid, which ultimately increases the transformation efficiency of Clostridium ljungdahlii to 1.73 ±0.04×10 ⁴ CFU/μg.

This is the highest efficiency reported so far for the electrotransformation of C. ljungdahlii pMTL83151 plasmid. While being highly efficient, this method also has the advantages of small bacterial collection volume and simple and fast production process, which is of great significance for the study of C. ljungdahlii.

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

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<213> Artificial sequence

<400> 5

tcccccgggt tggagattca aaaggtatct g 31

<210> 6

<211> 37

<212> DNA

<213> Artificial sequence

<400> 6

acgcgtcgac ttattcaact ttaagtacat ttttata 37

<210> 7

<211> 30

<212> DNA

<213> Artificial sequence

<400> 7

tcccccggga tgcataagtg gcgtgattta 30

<210> 8

<211> 42

<212> DNA

<213> Artificial sequence

<400> 8

acgcgtcgac ttaattattt tcaatatcat ttaatataat tc 42

<210> 9

<211> 36

<212> DNA

<213> Artificial sequence

<400> 9

tcccccgggt tggttatatttc tttaaaatgc attaag 36

<210> 10

<211> 31

<212> DNA

<213> Artificial sequence

<400> 10

acgcgtcgac tcatattttg cacagcatct c 31

<210> 11

<211> 34

<212> DNA

<213> Artificial sequence

<400> 11

tcccccggga tgaatagttt tataggttgg atag 34

<210> 12

<211> 36

<212> DNA

<213> Artificial sequence

<400> 12

acgcgtcgac tcaataattt ctaataatca attcac 36

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence

<400> 13

tcccccggga tggataaatc cgcaattaaa 30

<210> 14

<211> 35

<212> DNA

<213> Artificial sequence

<400> 14

acgcgtcgac ctatattttt gccaataaat tagcc 35

<210> 15

<211> 34

<212> DNA

<213> Artificial sequence

<400> 15

tcccccggga tgaacagttt tattgaagat gttg 34

<210> 16

<211> 37

<212> DNA

<213> Artificial sequence

<400> 16

acgcgtcgac tcagcaatta tcttttatct tttctac 37

Claims (28)

1. The application of the clostridium Yankeei self-restriction modification system is characterized in that the system is used for methylation modification of exogenous DNA of clostridium Yankeei so as to improve the electric conversion efficiency of the methylation modified exogenous DNA to clostridium Yankeei;

wherein, the clostridium Yankeei self-restriction modification system comprises a methylase coding gene CLJU_c24700;

the clostridium Yankee is selected from clostridium Yankee DSM 13528.

2. The use of claim 1, wherein the exogenous DNA comprises plasmid pMTL83151 and other sources of DNA sequences that are subsequently integrated into pMTL83151.

3. Use according to claim 1, characterized in thatThus, the electric conversion efficiency can be improved to be more than or equal to 10 ⁴ CFU/μg。

4. The process according to claim 3, wherein the electrical conversion efficiency is increased to at least 1.73X10 ⁴ CFU/μg。

5. A method for improving the electrical conversion efficiency of exogenous DNA of clostridium yang, comprising the steps of:

(a) Providing an exogenous DNA subjected to methylation modification by a clostridium Yankeei self-restriction modification system, wherein the clostridium Yankeei self-restriction modification system comprises a methylase coding gene CLJU_c24700;

(b) Electrotransformation of the exogenous DNA provided in step (a) into clostridium Yankee;

(c) Cell resuscitation of the clostridium Yankee;

the clostridium Yankee is selected from clostridium Yankee DSM 13528.

6. The method of claim 5, wherein in step (a) the methylation modification is accomplished in a modified plasmid host, the modified plasmid host being e.

7. The method of claim 6, wherein in step (a), the modified plasmid host is E.coli JM110, which has genotypedam ^- ，dcm ^- 。

8. The method of claim 5, wherein in the step (b), comprising the steps of: (b1) And adding a weakening agent into the clostridium Yankee thallus to weaken the cell wall of the clostridium Yankee.

9. The method of claim 8, wherein the weakening is performed when the cells are grown to an OD600 of 0.35 to 0.55.

10. The method of claim 9, wherein the weakening is performed when the cells are grown to an OD600 of 0.4 to 0.5.

11. The method of claim 8, wherein the attenuation agent is glycine at a final concentration of 0.5-5% w/v.

12. The method of claim 11, wherein the final concentration is 0.8-2.5% w/v.

13. The method of claim 11, wherein the final concentration is 1-1.5% w/v.

14. The method of claim 11, wherein the final concentration is 1.25% w/v.

15. The method of claim 8, further comprising, in step (b 1), adding a buffer protectant.

16. The method of claim 15, wherein the buffer protectant is sucrose.

17. The method of claim 5, wherein in step (b), the shock voltage is 500-1500V during the electrical conversion.

18. The method of claim 17, wherein the shock voltage is 800-1200V.

19. The method of claim 17, wherein the shock voltage is 1000V.

20. The method of claim 5, wherein in step (b), the shock capacitance is 30-100 μf in the electrical conversion.

21. The method of claim 20, wherein the shock capacitance is 40-60 μf.

22. The method of claim 20, wherein the shock capacitance is 50 μf.

23. The method of claim 5, wherein in step (b), the shock resistance is 120-300 Ω in the electrical conversion.

24. The method of claim 23, wherein the shock resistance is 150-250 Ω.

25. The method of claim 23, wherein the shock resistance is 200Ω.

26. The method of claim 5, wherein in step (c), the time for cell resuscitation is 15 to 25 hours.

27. The method of claim 26, wherein the time for cell resuscitation is 18 to 22 hours.

28. The method of claim 26, wherein the time for cell resuscitation is 20 hours.