CN105219795A - A kind of colibacillary method of ultrasonic assistant gene transformation - Google Patents

Abstract

The invention discloses a method for ultrasonically assisted exogenous gene transformation of Escherichia coli. The method comprises: mixing an expression plasmid carrying an exogenous gene with Escherichia coli competent cell suspension, adjusting the pH value to 6.5-7.5, The transformation reaction is carried out under the conditions of ~37°C and ultrasonic field strength of 200 ~ 500W. After the transformation reaction is completed, positive transformants of E. coli containing exogenous genes are screened; the E. coli competent cell suspension is obtained by E. coli. mixed with dimethyl sulfoxide; the ultrasound-induced gene transformation method of the present invention is more environmentally friendly, does not depend on the cell type, and the transformation rate is equivalent to the electric transformation rate, and the transformation rate reaches 2.6×10 ⁴ positive transformants/ μg of plasmid DNA, the enzyme activity of the recombinant strain with the highest yield reached 1.22U/mL, which was 144% higher than that of the original strain.

Description

A method for transforming Escherichia coli with exogenous gene assisted by ultrasound

(1) Technical field

The invention relates to a method for introducing exogenous genes into microorganisms, in particular to a high-efficiency ultrasonic transformation method for introducing constructed plasmids containing exogenous genes into Escherichia coli.

(2) Background technology

Cholesterol is widely present in animals, especially in brain and nerve tissue, and is also high in kidney, spleen, skin, liver and bile. Its solubility is similar to that of fat, insoluble in water, and easily soluble in solvents such as ether and chloroform. Cholesterol is an indispensable and important substance in animal tissue cells. It not only participates in the formation of cell membranes, but also is a raw material for the synthesis of bile acids, vitamin D and steroid hormones. Cholesterol can also be converted into bile acid, steroid hormones, and 7-dehydrocholesterol after metabolism, and 7-dehydrocholesterol can be converted into vitamin D3 after ultraviolet radiation, so cholesterol is not a harmful substance to the human body. But when it is excessive, it will cause hypercholesterolemia, which will have adverse effects on the body. Modern research has found that atherosclerosis, venous thrombosis, cholelithiasis and hypercholesterolemia are closely related. Cholesterol oxidase can oxidize cholesterol to cholest-4-en-3-one. Using the oxidizing property of cholesterol oxidase, a detection system for enzymatic detection of cholesterol content can be established. In addition, the oxidized product of cholesterol, cholest-4-en-3-one, has the effects of preventing heart disease, inhibiting skin keratinization, treating liver disease and preventing obesity. Therefore, cholesterol oxidase is an enzyme with various functions and uses, which is mainly derived from microorganisms.

The enzyme production ability of wild bacteria is about 300U/L, which cannot meet the requirements of industrial production. Therefore, scholars have been transforming the strains and optimizing the fermentation conditions to improve the enzyme production activity of the strains, the separation, purification and application of cholesterol oxidase. At present, they are all focused on improving the production of cholesterol oxidase through genetic engineering methods. Conventional genetic engineering methods require high-end instruments and equipment, and the reagents used are expensive and most of them are harmful to the human body and the environment. Ultrasound, as a physical method, can solve this disadvantage.

Ultrasonic technology is recognized as one of the high-tech and future industries in academia and engineering circles. In recent years, this high-tech has been obtained along with the great progress of physics, machinery, electronics, materials science and the needs of many marginal or cross-industrial fields. Rapid development. Since the 1980s, ultrasonic technology has been continuously applied in the fields of industry, agriculture, medicine and health. The application of ultrasonic technology in the field of biology has involved the crushing of cells, sterilization, extraction of intracellular substances, the use of micro-ultrasonic waves to enhance the production of intracellular enzymes and improve the homogeneous effect of non-aqueous phase enzyme catalysis and improve the wall membrane structure of cells , increase permeability and online detection of solid or liquid concentration in liquid, etc. Appropriate control of the intensity and frequency of ultrasonic action can have a positive effect on industrial microorganisms and improve the efficiency of industrial microbial fermentation processes. Ultrasonic transformation is an efficient method for introducing foreign genes into hosts. It is currently widely used in mammals and plants, but rarely reported in microorganisms.

(3) Contents of the invention

The invention provides a method for transforming Escherichia coli assisted by an ultrasonic wave, which is novel, mild in transformation conditions, low in cost, green in environment, and easy to realize gene transformation, and solves the problem of low transformation rate and low cell viability in the existing method problems such as death.

The present invention adopts following technical scheme:

The invention provides a method for ultrasonically assisted transformation of Escherichia coli with an exogenous gene. The method is as follows: mixing an expression plasmid carrying an exogenous gene with an Escherichia coli competent cell suspension, adjusting the pH value to 6.5-7.5, The transformation reaction is carried out under the conditions of 37°C and ultrasonic field strength of 200-500W. After the transformation reaction is completed, positive transformants of Escherichia coli containing exogenous genes are screened; Cells were mixed with dimethyl sulfoxide.

Further, the content of wet bacterial cells in the Escherichia coli competent cell suspension is 1×10 ⁷ -1×10 ⁸ cells/mL, and the final volume concentration of dimethyl sulfoxide is 1-4%.

Further, the conversion reaction is carried out at 35-37° C. and an ultrasonic field strength of 200-500 W for 10-30 minutes.

Further, the foreign gene in the expression plasmid carrying the foreign gene is cholesterol oxidase gene, and the nucleotide sequence is shown in SEQ ID NO.1.

atgaccgatagccgggcgaacagagccgatgcgactcggggggttgcatccgtctcacgccgtcgattccttgcgggcgcaggtctgaccgcgggagccatcgcgctctcgtcgatgtcgacctccgcctccgcagcccccagccgcaccctcgccgacggcgaccgcgtccctgccctcgtcatcggcagtggatacggcggtgccgtcgccgcgctgcggctgacgcaggccggtatccccacgcagatcgtcgagatgggccgcagctgggacaccccgggctccgacggcaagatcttctgcgggatgctcaaccccgacaagcgctcgatgcggttggccgacaagaccgatcagccggtcagcaacttcatgggcttcggcatcaacaagagcatcgaccggtacgtcggcgtcctcgactccgagcggttctccggcatcaaggtctaccagggccgcggcgtcggcggcggctcgctcgtcaacggcggtatggcagtcaccccgaagcgcaactacttcgaggagatcctgccgtcggtcgactcgaacgagatgtacaacaagtacttcccgcgcgccaacaccggtctgggtgtcaacaacatcgaccaggcgtggttcgagtccaccgagtggtacaagttcgcccgcaccggccgcaagaccgcccaacgttcgggtttcacaaccgctttcgtgcccaacgtgtacgacttcgagtacatgaagaaggaggctgccggccaggtcaccaagtcgggcctcggcggtgaggtcatctacggcaacaacgccggcaagaagtcgctcgacaagacctacctcgcgcaggccgcggccaccgggaagctgacgatcacgactttgcaccgcgtcaccaaggtcgcgccggccaccggcagcggctacagcgtgacgatggaacagatcgacgagcagggcaacgtcgtcgccaccaaggtcgtcaccgccgatcgggtgttcttcgcgg ccggcagcgtcggcaccagcaagctcctggtctcgatgaaggcgcagggccacctgccgaacctgtcgtcgcaagtcggcgagggctggggcaacaacggcaacatcatggtgggccgcgcgaaccacatgtgggacgccaccggatccaagcaggccaccatcccgacgatgggaatcgacaactgggccgacccggcggcaccgatcttcgcggagatcgccccgctgccggccgggctcgagacctacgtcagcctgtacctggccatcacgaagaaccccgaacgtgctcgcttccagttcaattcgggcaccggcaaggtcgatctcacctgggctcagtcgcagaaccagaagggcatcgacatggccaagaaggtgttcgacaagatcaaccagaaggaaggcacgatctaccggaccgatctgttcggcgtgtacaagacgtggggcgacgacttcacgtaccacccgctgggcggcgtgctgctgaacaaggcgaccgacaacttcggccgcctgcccgagtaccccgggctgtacgtggtggacggctcgctcgtccccggcaatgtcggcgtcaacccgttcgtcacgatcaccgcgctcgccgagcgcaacatggacaagatcatctcgtccgacatccagtga。

Further, the Escherichia coli is Escherichia coli E.coli (DE3), purchased from Novagen.

Further, the volume ratio of the expression plasmid carrying the exogenous gene to the Escherichia coli competent cell suspension is 1:40-55, and the content of wet bacterial cells in the Escherichia coli competent cell suspension is 1×10 ⁷ ~1 ×10 ⁸ cells/mL.

Further, the Escherichia coli competent cells are prepared according to the following steps: inoculate Escherichia coli into LB liquid medium, 180-220r/min, shake culture at 35-37°C until exponential growth phase (usually 2.5-10h), 12000rr /min, 10-15min, centrifuge at 4°C, collect the wet cells, and obtain Escherichia coli competent cells.

Further, the expression plasmid carrying the cholesterol oxidase gene is a pET28a-choB vector plasmid.

The method for ultrasonic-assisted exogenous gene transformation Escherichia coli described in the present invention is specifically carried out as follows:

(1) Cultivation and collection of Escherichia coli competent cells

Pick a single colony of Escherichia coli (preferably Escherichia coli E.coli (DE3)), inoculate it into LB liquid medium, and cultivate it with shaking at 180-220r/min, 35-37°C until the exponential growth phase (usually 2.5-20h), 12000r/min, 10-15min, centrifuge at 4°C, collect the wet cells, and obtain Escherichia coli competent cells;

(2) Gene transformation research using Escherichia coli as host

Add dimethyl sulfoxide to E. coli competent cells so that the content of wet bacterial cells is 1×10 ⁷ ~1×10 ⁸ cells/mL (the final volume concentration of dimethyl sulfoxide is 1-4%), add The pET28a carrier plasmid carrying the cholesterol oxidase gene was transformed by ultrasonic waves at a pH value of 6.5-7.5, 35-37°C, and an ultrasonic field strength of 200-500W. The effect of parameters such as field strength and treatment time on the transformation influences;

(3) Screening of positive transformants

When the transformation is completed, spread the transformed bacterial solution onto an LB plate containing 50-100 μg/mL Kan and 50-100 μg/mL, and culture at 35-37°C for 24-48 hours for screening to obtain positive transformants;

(4) Identification of positive transformants

Randomly select transformants on the transformation plate to extract genomic DNA, use it as a template, and use specific primers for PCR verification. Primers used: 5'-TTCCATGGCCGATAGCCGGGCGAACAG-3' and 5'-GCGAATTCTCACTGGATGTCGGACGAGATGA-3'. The PCR reaction conditions were: 94°C for 5min; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 5min; gel electrophoresis was used to detect whether the recombinant vector was inserted into the E. coli genome.

(5) Product expression and stability research

The identified transformants were inoculated into LB liquid medium containing 50-100 μg/mL Kan and 50-100 μg/mL, cultured at 35-37°C for 12-15 hours, and then added IPTG to a final concentration of 0.1-0.6 mmol/L , continue culturing for 3-5 hours, and detect the enzyme activity of cholesterol oxidase.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

1) At present, most reports focus on ultrasound-induced gene transformation of plants and animals. The present invention intends to use ultrasound to induce microbial gene transformation.

2) Commonly used gene transformation methods include genetic engineering and electroporation. Genetic engineering requires high-end instruments and equipment, and the reagents used are expensive and mostly harmful to the human body and the environment; electroporation requires lower ionic media and higher voltage , and conjugation requires direct cell-cell contact. The ultrasonic-induced gene transformation method of the present invention is more environmentally friendly, does not depend on the cell type, and the transformation rate is equivalent to the electric transformation rate. Compared with the existing electrotransformation method, the present invention has the above-mentioned advantages, and the transformation efficiency is also equivalent to or higher than that of electrotransformation. For example, the transformation rate of an electrotransformation method (CN1718732A) that introduces a shuttle plasmid into Brevibacterium flavum is 2.0×10 ³ , the conversion rate of the present invention reaches 2.6×10 ⁴ positive transformants/μg plasmid DNA, and the enzyme activity of the recombinant strain with the highest yield reaches 1.22U/mL, which is 144% higher than that of the original strain.

(4) Description of drawings

Fig. 1 is the gel electrophoresis image of positive transformant screening in Example 1, swimming lane M is Marker, and swimming lane 1 is positive transformant.

Fig. 2 is the gel electrophoresis image of positive transformant screening in Example 2, swimming lane M is Marker, and swimming lane 1 is positive transformant.

Fig. 3 is the gel electrophoresis image of positive transformant screening in Example 3, swimming lane M is Marker, and swimming lane 1 is positive transformant.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

The formula of LB liquid medium is: tryptone 10g, yeast extract 5g, NaCl 10g, dilute to 1L with deionized water.

Example 1

(1) Cultivation and collection of Escherichia coli competent cells

Pick Escherichia coli E.coli (DE3) (purchased from Novagen) and inoculate it into LB medium, shake at 180r/min at 35°C for 2.5h to the exponential growth phase, centrifuge at 12000r/min for 10min at 4°C, and collect wet bacteria To obtain Escherichia coli competent cells.

(2) Gene transformation research using Escherichia coli as host

The cholesterol oxidase gene choB was cloned into pET28a vector plasmid.

The wet thallus obtained in step (1) is mixed with dimethyl sulfoxide to make a thalline concentration of ¹ × 108 cells/mL, and the final concentration of dimethyl sulfoxide is 2% of the cell suspension. The pET28a plasmid of the cholesterol oxidase gene choB (plasmid concentration should not be less than 70ng/μL) was mixed with the cell suspension at a volume ratio of 1:40, that is, 0.5mL of the plasmid was mixed with 20mL of the cell suspension, and the pH was adjusted to 6.5; To 35°C, the ultrasonic field strength is 200W, and the processing time is 15min.

(3) Screening and identification of positive transformants

Screening of positive transformants: spread the transformed bacterial solution on an LB plate containing 50 μg/mLKan and 50 μg/mLCam, incubate at 35°C for 24 hours, perform plate screening, and obtain positive transformants.

Verification of positive transformants: randomly select transformants on the transformation plate to extract genomic DNA, use it as a template, and use specific primers for PCR verification. The PCR reaction conditions are: 94°C for 5min; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 5min. Gel electrophoresis was used to detect whether the recombinant vector was inserted into the E. coli genome, and the results are shown in Figure 1.

Primers used: 5'-TTCCATGGCCGATAGCCGGGCGAACAG-3' and 5'-GCGAATTCTCACTGGATGTCGGACGAGATGA-3'.

At the same time, a negative control was set up for ultrasonic transformation. Except that no plasmid DNA was added to the negative control, the rest of the process was exactly the same as other treatments. In the case of no colony growth in the negative control on the LB plate, count the number of transformants and calculate the transformation efficiency.

Transformation efficiency = number of transformants/amount of plasmid DNA added (μg) × dilution factor.

(4) Product expression

The identified positive transformants were inoculated into LB liquid medium containing 50 μg/mLKan and 50 μg/mLCam, cultured at 35°C for 12 hours to reach the logarithmic growth phase, then added IPTG to a final concentration of 0.2mmol/L, and continued at 35°C After culturing for 3 hours, the enzyme activity of cholesterol oxidase was detected (enzyme activity is defined as: the amount of enzyme that converts 1 μmol of cholesterol into cholest-4-en-3-one in 1 minute at 37°C is defined as 1 enzyme activity unit (U)).

During the above fermentation process, the obtained fermentation broth was analyzed for enzyme activity, and the conversion rate was 1.3×10 ⁴ positive transformants/μg plasmid DNA, and the enzyme activity of the recombinant strain with the highest yield (ie Escherichia coli containing the cholesterol oxidase gene) was It reached 1.12U/mL, an increase of 124% compared with the starting strain.

Example 2

(1) Cultivation and collection of Escherichia coli competent cells

Pick Escherichia coli E.coli (DE3) (purchased from Novagen) and inoculate it into LB medium, culture with shaking at 200r/min at 36°C for 20h to the late stage of exponential growth, centrifuge at 12000r/min for 10min at 4°C, and collect the wet cells , to obtain Escherichia coli competent cells.

(2) Gene transformation research using Escherichia coli as host

The cholesterol oxidase gene choB was cloned into pET28a vector plasmid.

The wet thallus obtained in step (1) is mixed with dimethyl sulfoxide to make a cell suspension with a cell content of 1×10 ⁸ cells/mL and a final volume concentration of dimethyl sulfoxide of 3%. The pET28a plasmid of the oxidase gene choB (plasmid concentration should not be less than 70ng/μL) was mixed with the cell suspension at a volume ratio of 1:50, that is, 0.5mL of the plasmid was mixed with 25mL of the cell suspension, and the pH was adjusted to 7.0; To 36°C, the ultrasonic field strength is 500W, and the treatment time is 30min.

(3) Screening and identification of positive transformants

Screening of positive transformants: spread the transformed bacterial solution on an LB plate containing 80 μg/mLKan and 80 μg/mLCam, incubate at 36°C for 36 hours, perform plate screening, and obtain positive transformants.

Verification of positive transformants: randomly select transformants on the transformation plate to extract genomic DNA, use it as a template, and use specific primers for PCR verification. The PCR reaction conditions are: 94°C for 5min; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 5min. Gel electrophoresis was used to detect whether the recombinant vector was inserted into the E. coli genome, and the results are shown in Figure 2.

Primers used: 5'-TTCCATGGCCGATAGCCGGGCGAACAG-3' and 5'-GCGAATTCTCACTGGATGTCGGACGAGATGA-3'.

At the same time, a negative control was set up for ultrasonic transformation. Except that no plasmid DNA was added to the negative control, the rest of the process was exactly the same as other treatments. In the case of no colony growth in the negative control on the LB plate, count the number of transformants and calculate the transformation efficiency.

Transformation efficiency = number of transformants/amount of plasmid DNA added (μg) × dilution factor.

(4) Product expression

The identified positive transformants were inoculated into LB liquid medium containing 50 μg/mLKan and 50 μg/mLCam, cultured at 36°C for 14 hours, added IPTG to a final concentration of 0.4mmol/L, continued to culture at 36°C for 4h, and detected cholesterol Oxidase activity.

During the above fermentation process, the obtained fermentation broth was analyzed for enzyme activity, and the conversion rate was 2.6×10 ⁴ positive transformants/μg plasmid DNA, and the enzyme activity of the recombinant strain with the highest yield reached 1.22 U/mL, which was higher than that of the original strain. Live increased by 144%.

Example 3

(1) Cultivation and collection of Escherichia coli competent cells

Pick Escherichia coli E.coli (DE3) (purchased from Novagen) and inoculate it into LB medium, shake it at 220r/min at 37°C for 8h, centrifuge at 12000r/min for 15min at 4°C, collect the wet cells, and obtain the large intestine Bacillus Competent Cells.

(2) Gene transformation research using Escherichia coli as host

The cholesterol oxidase gene choB was cloned into pET28a vector plasmid.

The wet thallus obtained in step (1) is mixed with dimethyl sulfoxide to make a cell suspension with a cell content of 1×10 ⁸ cells/mL, and a final volume concentration of dimethyl sulfoxide of 4%. Mix the pET28a plasmid of the oxidase gene choB (plasmid concentration should not be less than 70ng/μL) with the cell suspension at a volume ratio of 1:55, that is, mix 0.5mL of the plasmid with 27.5mL of the cell suspension, and adjust the pH to 7.5; Adjust the temperature to 37°C, the ultrasonic field strength is 500W, and the treatment time is 30min.

(3) Screening and identification of positive transformants

Screening of positive transformants: spread the transformed bacterial solution onto an LB plate containing 100 μg/mLKan and 100 μg/mLCam, incubate at 37°C for 48 hours, perform plate screening, and obtain positive transformants.

Verification of positive transformants: randomly select transformants on the transformation plate to extract genomic DNA, use it as a template, and use specific primers for PCR verification. The PCR reaction conditions are: 94°C for 5min; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 5min. Gel electrophoresis was used to detect whether the recombinant vector was inserted into the E. coli genome, and the results are shown in Figure 3.

Primers used: 5'-TTCCATGGCCGATAGCCGGGCGAACAG-3' and 5'-GCGAATTCTCACTGGATGTCGGACGAGATGA-3'.

At the same time, a negative control was set up for ultrasonic transformation. Except that no plasmid DNA was added to the negative control, the rest of the process was exactly the same as other treatments. In the case of no colony growth in the negative control on the LB plate, count the number of transformants and calculate the transformation efficiency.

Transformation efficiency = number of transformants/amount of plasmid DNA added (μg) × dilution factor.

(4) Product expression

The identified positive transformants were inoculated into LB liquid medium containing 100 μg/mLKan and 100 μg/mLCam, cultured at 37°C for 15 hours to reach the logarithmic growth phase, added IPTG to a final concentration of 1.2mmol/L, and continued at 37°C After culturing for 5 hours, the enzyme activity of cholesterol oxidase was detected.

During the above fermentation process, the obtained fermentation broth was analyzed for enzyme activity, and the conversion rate was 9.2×10 ³ positive transformants/μg plasmid DNA, and the enzyme activity of the recombinant strain with the highest yield reached 1.16 U/mL, which was higher than that of the original strain. Live increased by 132%.

Claims (8)

1. A method for ultrasound-assisted exogenous gene transformation Escherichia coli, characterized in that, the method is: mixing the expression plasmid carrying the exogenous gene with Escherichia coli competent cell suspension, adjusting the pH value to 6.5～7.5, The transformation reaction is carried out under the conditions of 35-37°C and an ultrasonic field strength of 200-500W. After the transformation reaction is completed, positive transformants of E. coli containing exogenous genes are screened; the E. coli competent cell suspension is prepared from Bacillus competent cells mixed with dimethyl sulfoxide. 2. The method of ultrasonic-assisted exogenous gene transformation of Escherichia coli according to claim 1, characterized in that the content of wet bacterial cells in the Escherichia coli competent cell suspension is 1×10 ⁷ to 1×10 ⁸ cells/ mL. 3. The method of ultrasonic-assisted exogenous gene transformation of Escherichia coli according to claim 1, characterized in that the transformation reaction is carried out at 35-37° C. and an ultrasonic field strength of 200-500 W for 10-30 minutes. 4. The method for ultrasonically assisted exogenous gene transformation of Escherichia coli as claimed in claim 1, characterized in that the exogenous gene in the expression plasmid carrying the exogenous gene is cholesterol oxidase gene. 5. The method for ultrasonically assisted exogenous gene transformation Escherichia coli as claimed in claim 1, characterized in that said Escherichia coli is Escherichia coli E.coli (DE3). 6. The method for ultrasonically assisted exogenous gene transformation E. coli as claimed in claim 1, characterized in that the volume ratio of the expression plasmid carrying the exogenous gene to the E. coli competent cell suspension is 1:40-55. 7. The method for transforming Escherichia coli with ultrasonic-assisted exogenous gene as claimed in claim 1, characterized in that said Escherichia coli competent cells are prepared according to the following steps: inoculate Escherichia coli into LB liquid medium, 180～220r/min , shake culture at 35-37°C to the exponential growth phase, centrifuge at 12000r/min for 10-15min at 4°C, collect the wet cells, and obtain Escherichia coli competent cells. 8. The method for ultrasonically assisted exogenous gene transformation of Escherichia coli as claimed in claim 1, characterized in that the expression plasmid carrying the cholesterol oxidase gene is a pET28a-choB vector plasmid.