TWI739270B - Method of preparing escherichia coli for producing phage and method of using escherichia coli for producing phage - Google Patents

Abstract

The present invention provides a method for preparing bacteriophage-producing Escherichia coli and the production of bacteriophage by the method Methods. The Escherichia coli strain has undergone genetic recombination to strengthen its sugar metabolism pathway, strengthen its electron transport chain pathway, inhibit its lpp gene expression or modify its transcription, thereby enhancing the energy charge state of Escherichia coli as the host strain to solve The host strain produces a large amount of phage shock protein, which leads to the problem of poor energy status of the host strain. The preparation method of the above-mentioned Escherichia coli is that the donor DNA that can produce the above-mentioned gene expression effect is transferred into Escherichia coli for gene recombination to prepare the aforementioned transgenic Escherichia coli. The above-mentioned method of producing phage is to provide the aforementioned transgenic Escherichia coli and make it infected with M13 phage. By the above-mentioned preparation method of Escherichia coli that produces phage and the method for producing phage, the problem that the ability of conventional M13 phage host bacteria to produce M13 phage is inhibited can be solved.

Description

Preparation method of bacteriophage-producing Escherichia coli and its production method method

The present invention relates to a method for preparing bacteriophage-producing Escherichia coli and a method for producing bacteriophages, especially an Escherichia coli used to produce M13 bacteriophage.

Phage is a protein expression carrier commonly used in molecular biotechnology. With the development of phage display technology, various foreign proteins can be further displayed on the surface of phage, and other substances can be combined with foreign proteins on the surface of phage to study the interaction between specific proteins and other substances. Combining or combining foreign proteins on the surface of phage with inorganic substances such as organic substances or metal ions to form nanomaterials.

Among them, the M13 bacteriophage is a piezoelectric bacteriophage, that is, the M13 bacteriophage generates electricity when pressed by an external force. Therefore, the M13 bacteriophage can be used to make batteries. On the other hand, M13 bacteriophage is also suitable for phage display platform technology to produce various types of peptides for the manufacture of various nanomaterials, such as lithium battery nanoelectronic materials, biofuel cell materials or It is a biosensor material, etc. Compared with the conventional nanomaterial manufacturing process, nanomaterials made with M13 bacteriophage have advantages in material cost, safety and process equipment cost.

The current method of producing M13 phage mainly uses Escherichia coli as a host and uses Escherichia coli to produce M13 phage. However, in the process of using E. coli to produce M13 phage, M13 phage uses E. coli to produce phage DNA and package the proteins required for phage DNA. Among them, the phage protein pIV forms a channel on the E. coli cell membrane to facilitate the release of phage. As a result, it will change the structure of the E. coli cell membrane and cause an imbalance of ions in the E. coli cell membrane, which will lead to pressure on the growth of the host E. coli. In response to the growth adversity caused by the M13 bacteriophage, the host Escherichia coli will express a large number of phage-shock proteins to maintain the growth state of Escherichia coli. Based on the above-mentioned mechanism of E. coli resistance to adversity, when the host E. coli is infected by M13 phage, it will not only consume a lot of energy to produce M13 phage DNA and protein, but also need to produce a large amount of phage shock protein to counter its growth adversity, resulting in the host large intestine. The poor energy status of the bacilli inhibits its growth and therefore also inhibits the ability of the host E. coli to produce phage.

The purpose of the present invention is to solve the above problems and provide a method for preparing Escherichia coli for the production of M13 bacteriophage, which comprises the following steps: (a) providing Escherichia coli; (b) transgenerating the donor DNA of the _{bacteriophage PL promoter} Into the Escherichia coli, the donor DNA can strengthen the sugar metabolism pathway of the Escherichia coli; and (c) the donor DNA is inserted into the chromosome of the Escherichia coli by genetic recombination.

In order to achieve the above and other objectives, the present invention provides a method for preparing Escherichia coli for the production of M13 phage, which comprises the following steps: (a) providing Escherichia coli; (b) transducing the donor DNA of the _{phage PL promoter} Colonized into the Escherichia coli, the donor DNA can strengthen the electron transport chain path of the Escherichia coli; and (c) the donor DNA is inserted into the chromosome of the Escherichia coli by genetic recombination.

To achieve the above and other objectives, the present invention provides a method for preparing Escherichia coli for the production of M13 phage, which comprises the following steps: (a) providing Escherichia coli; and (b) inserting an internal kanamycin-resistant gene The specific DNA fragment of the lpp gene of E. coli is inserted into the lpp gene of E. coli, thereby inhibiting the lpp gene of E. coli.

To achieve the above and other objectives, the present invention provides a method for preparing Escherichia coli for the production of M13 bacteriophage, which comprises the following steps: (a) providing Escherichia coli; (b) translating the mutant rpoS gene or the mutant rpoD gene into In the Escherichia coli; and (c) the mutant rpoS gene or the mutant rpoD is transformed into the Escherichia coli in a plastid form, thereby changing the transcription in the Escherichia coli.

To achieve the above and other objectives, the present invention provides a method for producing M13 phage, which comprises the following steps: (a) providing Escherichia coli prepared by the method according to any one of claims 1 to 5; and ( b) Infect the Escherichia coli with M13 phage.

With the above-mentioned phage-producing strain, its preparation method, and its phage production method, the problem that the ability of the conventional M13 phage host bacteria to produce M13 phage is inhibited can be solved.

Fig. 1 is a schematic diagram of the central metabolic pathway of Escherichia coli; Fig. 2 is a diagram of the experimental results of a comparison experiment of the growth status and phage yield of the infected transgenic strain and the proto-strain of Example 1 of the present invention; Fig. 3 is a graph of the experimental results of the growth status and phage yield comparison experiment between the infected transgenic strain and the original strain of Example 2 of the present invention; Fig. 4 is the growth status of the infected transgenic strain and the original strain of Example 3 of the present invention And Fig. 5 is a graph showing the growth status of the infected transgenic strain and the original strain and the experimental result graph of the phage production comparison experiment of Example 4 of the present invention.

In order to fully understand the purpose, features and effects of the present invention, the following specific examples and accompanying drawings are used to explain the present invention in detail. The description is as follows: Host strain cultivation method: here cultivation method Among them, Escherichia coli strain ER2738 was selected as the host strain of M13 phage. Escherichia coli strain ER2738 was obtained by New England Biolabs. The cultivation process of strain ER2738 is as follows.

Firstly, the strain ER2738 was cultured in Luria-Bertani (LB) liquid medium (containing 20g/ml tetracycline) at 37°C. After the cells were cultured, _{the absorbance value of OD 550} was measured by a spectrophotometer of 3~5. At this time, take 700 μL of the aforementioned cultured ER2738 bacterial liquid into a sterile cryotube, and add 300 μL of 50% (v/v) sterile glycerin to the sterile cryotube to mix, and then place the frozen tube containing the ER2738 bacterial liquid into the sterile tube. Store in -80℃ freezer. When the strain ER2738 is to be used, the frozen tube containing the ER2738 bacterial liquid is taken out of the -80℃ freezer for dissolution, and the bacterial liquid of the bacterial strain ER2738 is taken out from the frozen tube, and painted on the aseptic LB solid medium. The LB solid medium with ER2738 bacterial solution is placed in a constant temperature incubator (the ambient temperature is set to 37°C). After the ER2738 colonies in the aforementioned LB solid medium grow to an appropriate size, a single colony (signal colony) of the ER2738 colony is taken to M9-YM In the liquid medium and add an appropriate amount of antibiotics (10g/ml tetracycline in this example), and then put it in a shaking incubator for shaking culture (the ambient temperature is set to 37°C), and the ER2738 colony cultured above is used as the follow-up For experimental use. The aforementioned M9-YM medium contains the following components: yeast extract (5g/L), NH ₂ HPO ₄ . 12H ₂ O (15.2g/L), KH ₂ PO ₄ (3g/L), NaCl (0.5g/L), NH ₄ Cl (1g/L), MgSO ₄ (1mM), CaCl ₂ (0.1mM), vitamin B1(0.01g/L), FeSO ₄ . 7H ₂ O (0.008g/L), Al ₂ (SO ₄ ) ₃ . 16H ₂ O (0.0013g/L), ZnSO ₄ . 7H ₂ O (0.0002g/L), CuCl ₂ . 2H ₂ O (0.0001g/L), NaMoO ₄ . 2H ₂ O (0.0002g/L), MnCl ₂ (0.0007g/L), CoCl ₂ . 6H ₂ O (0.00007g/L), H ₃ BO ₃ (0.00005g/L).

Host strain gene recombination method: In this recombination method, in order to prepare a host strain ER2738 that can produce phage in high quantities, the λ Red homologous recombination system (λ Red recombination system) is used to recombine the genes of strain ER2738 to prepare The required transgenic strain ER2738 was produced. The process of gene recombination of the host strain ER2738 gene by using the lambda Red homologous recombination system is as follows.

First, the strain ER2738 was obtained by the aforementioned host strain culture method, and then the pKD46 plastid containing the gam, bet, and exo genes of the lambda phage was sent into the strain ER2738 by the DNA transformation method, so that the strain ER2738 could be produced separately The proteins Gam, Bet, and Exo expressed by the gam, bet, and exo genes of λ phage are used to assist the linear donor DNA (donor DNA) transferred into the strain ER2738 through DNA transformation. Homologous recombination with the gene of strain ER2738. Gam protein can inhibit the enzymatic activity of RecBCD exonuclease V in E. coli cells, so that RecBCD exonuclease V cannot decompose the linear donor DNA transferred through DNA transformation. EXO protein can decompose double strand DNA (dsDNA) form Donor DNA, leaving the donor DNA in the form of single strand DNA (ssDNA). Beta protein binds to the donor DNA in the form of single-stranded DNA, and promotes the homologous recombination reaction between the donor DNA and the gene of strain ER2738. Through the effects of the aforementioned proteins Gam, Bet and Exo, the homologous recombination reaction between the donor DNA and the strain ER2738 gene can be completed, thereby preparing the desired transgenic strain ER2738.

In the λ Red homologous recombination system, in order to allow the donor DNA and the gene of strain ER2738 to undergo homologous recombination, and to screen out the transgenic strain ER2738 that successfully completed the gene recombination, the head and tail ends of the donor DNA sequence It needs to have a homologous base pairing sequence with the target gene sequence (that is, the sequence that undergoes homologous recombination with the donor DNA), and the donor DNA contains an antibiotic resistance gene to facilitate subsequent screening.

The aforementioned pKD46 plastids used to assist homologous gene recombination are used to regulate the expression of gam, bet and exo genes on the pKD46 plastids through the Arabinose operon. Since pKD46 plastids are heat-sensitive plastids, they are unstable at high temperatures (about 42°C). After homologous gene recombination is completed with pKD46 plastids, the heat-sensitive properties of pKD46 plastids can be used to reduce The pKD46 plastids in the transgenic strain ER2738 were removed to stop the pKD46 plastids and continue to show the proteins Gam, Bet and Exo that are not needed in the subsequent experiments. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci US A.2000 Jun 6; 97(12): 6640-5 .

After screening the required transgenic strain ER2738, Cre recombinase is further used to remove the antibiotic resistance gene in the transgenic strain ER2738. In this recombination method, the pCP20 plastid is transferred into the transgenic strain ER2738 to express Cre recombinase. Since the origin of replication repA101ts of pCP20 plastids is thermosensitive and unstable under high temperature environments, pCP20 plastids can be used to remove antibiotic resistance genes in the transgenic strain ER2738. The heat-sensitive characteristics of the body eliminate it. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci US A. 2000 Jun 6; 97(12): 6640-5 .

DNA transformation method: firstly prepare competent cells of strain ER2738, and then use the electric shock penetration method to enable the aforementioned donor DNA (donor DNA) and plastids to enter strain ER2738 for genetic recombination.

Preparation of competent cells of strain ER2738: First, the strain ER2738 obtained by the aforementioned host strain culture method was cultured on a solid medium, and then a single colony of strain ER2738 was taken from the solid medium to LB culture medium and an appropriate amount of antibiotics (in this example 20g/ml tetracycline) incubate at a suitable temperature for 12 to 16 hours. Next, take 5ml of the aforementioned LB culture broth containing the strain ER2738 for transformation, adjust the initial cell concentration in the LB broth containing the strain ER2738 to 0.08 (OD550), and inoculate 500ml of the fresh LB broth into the shaking incubator In the cultivation. When the cell density of the strain ER2738 culture solution reaches 0.3-0.5, take the strain ER2738 culture solution out of the incubator and ice-bath for 5 minutes, and then centrifuge the strain ER2738 culture solution at 4000 rpm for 2 minutes. After the centrifugation is complete, pour it out. The clear liquid leaves the precipitate of strain ER2738. Mix the strain ER2738 precipitate with 5ml of 10% glycerol after ice bath for washing step. The mixture of strain ER2738 precipitate and 10% glycerol is centrifuged at 4000rpm for 10 minutes. After centrifugation, the supernatant is discarded and the strain is left. The precipitate of ER2738. After the above washing steps were repeated three times, the precipitate of strain ER2738 was re-dissolved with an appropriate amount of 10% glycerol after an ice bath to complete the preparation of competent cells of strain ER2738.

Electric shock penetration method: 40μl of competent cells of strain ER2738 (obtained through the aforementioned preparation method) and 2μl of purified plasmid or linear donor DNA fragments are mixed and ice-bathed for 1 minute, and then the competent cells are combined with the plasmid or The mixture of linear donor DNA fragments is transferred to a 2mm electroporation tube, and the aforementioned mixture is shocked with a voltage of 2500 volts. In this way, the plastid or linear donor DNA fragments can be transferred into the strain ER2738, and the subsequent strains will continue. The ER2738 gene was recombined to obtain the transgenic strain ER2738.

After the electric shock is completed, add 1 ml of ice-bathed SOC culture solution to the aforementioned mixture, and place it in a shaking incubator for shaking culture at a temperature of 37°C. After shaking and incubating the aforementioned mixture containing SOC medium for about 2 to 3 hours, then take out the aforementioned mixture containing SOC medium and centrifuge at 4000 rpm for 10 minutes. After centrifugation, the supernatant is discarded and the colonization is left. Bacteria of strain ER2738, which can be used for subsequent experiments.

DNA extraction and purification methods: DNA extraction uses the Blood & Tissue Genomic DNA Extraction Miniprep System set produced by VIOGENE for strain chromosome extraction. The applicable scope of the Blood & Tissue Genomic DNA Extraction Miniprep System set includes Gram-positive bacteria. (Gram-positive bacteria) and Gram-negative bacteria (Grame-ngative bacteria). Plasmid extraction uses FavorPrep ^TM Plasmid DNA Extraction Mini Kit. The DNA purification system uses the Gel/PCR DNA Fragments Extraction Kit produced by Geneaid for DNA purification.

Virus plaque counting method: First, provide the bacterial solution of the host strain ER2738 infected with M13 bacteriophage, centrifuge the bacterial solution containing the virus at 12000 rpm for 10 minutes, and collect the supernatant. Dilute the virus-containing supernatant by 10 ⁴ to 10 ^{9 to} prepare a dilution. Take 10μl of the diluted solution and 100μl of fresh bacterial solution and mix for five minutes, then add 3ml of semi-solid gel (soft agar) with a temperature of about 45℃ and mix well to form a mixed solution. Pour the aforementioned mixed solution containing 5-bromo-4 -Chloro-3-indolyl-β-D-galactopyranoside (X-gal) on the plate medium. After the plate medium poured into the mixed solution is allowed to stand overnight at 37°C, blue-green colonies can be observed on the plate medium. The aforementioned blue-green colonies represent virus plaques. The number of virus plaques is calculated and multiplied by The dilution factor can get the phage titer (unit: pfu/mL).

Example 1. Experiment on the sugar metabolism pathway of the enhanced strain ER2738: In this Example 1, the problem of inhibiting the ability of the host bacteria to produce M13 phage was solved by preparing a transgenic strain ER2738-1 that enhances the sugar metabolism pathway of the strain ER2738.

The mechanism to solve the problem that the host bacteria's ability to produce M13 phage is inhibited by strengthening the host strain’s sugar metabolism pathway is as follows: The health state of the host cell can be described by the energy charge. Generally speaking, it is in normal growth. The host cell under the state (energy charge={[ATP]+1/2[ADP]}/{[ATP]+[ADP]+[AMP]}), its energy charge can reach 0.8 or more, as for the death of the host cell The energy charge will be reduced to less than 0.5, which indicates that healthy host cells have a better energy status. Therefore, by modifying the sugar metabolism pathway of the host strain E. The strain has more energy to produce the DNA and protein of the M13 phage and resist the growth adversity of the host strain, thereby enhancing the host bacteria's ability to produce M13 phage.

In this example 1, the pentose phosphate pathway, glycolysis pathway, and the conversion of NADPH to NADH pathway of the transgenic strain ER2738-1 were enhanced to enhance the transgenic strain ER2738. -1 energy production value.

Figure 1 shows the pentose phosphate pathway in E. coli cells. The pentose phosphate pathway is the main source of NADPH in the cell, and NADPH is an important energy molecule in the cell.

Figure 1 also shows the glycohydrolysis pathway in E. coli cells. The end product of the glycohydrolysis pathway is pyruvate. Continued oxidation of pyruvate can introduce the metabolic carbon stream into the citric acid cycle pathway for further gains. NADPH and ATP.

Figure 1 also shows the conversion pathway of NADPH in E. coli cells into NADH. It can be seen from the figure that NADPH is converted into NADH through the transhydrogenase expressed by the udhA gene.

In this example 1, by enhancing the expression of the pentose phosphate pathway zwf gene of the transgenic strain ER2738-1, thereby increasing the NADPH production ability of the transgenic strain ER2738-1; by enhancing the aceEF in the glycohydrolysis pathway of the transgenic strain ER2738-1 Gene expression, and insert the mutant lpdA gene into the transgenic strain ER2738-1 to accelerate the conversion of pyruvate into acetyl-CoA (acetyl-CoA). Acetyl-CoA enters the citric acid cycle to produce NADH and ATP again; Enhance the expression of udhA gene of transgenic strain ER2738-1 to increase NADH production and increase the energy charge of transgenic strain ER2738-1.

The preparation process of the transgenic strain ER2738-1 is as follows.

In Example 1, _{the donor DNA of the following bacteriophage PL} promoter was sent to strain ER2738 for genetic recombination: the donor DNA that can enhance the expression of the pentose phosphate pathway zwf gene of E. coli; can enhance the glycohydrolysis pathway of E. coli Donor DNA for the expression of the aceEF gene in aceEF; Donor DNA for the mutant lpdA gene used to accelerate the conversion of pyruvate into acetyl coenzyme; Donor DNA for the expression of the udhA gene in Escherichia coli.

In this example 1, the donor DNA that enhances the expression of zwf gene, the donor DNA that enhances the expression of aceEF gene, and the donor DNA that enhances the expression of udhA gene are inserted into the transgenic strain through the aforementioned DNA transformation method and host strain gene recombination method ER2738-1 within the chromosome. However , the insertion method of the donor DNA of the mutant lpdA gene does not use the aforementioned λ Red homologous recombination system. The process of inserting the donor DNA of the mutant lpdA gene into the chromosome of the transgenic strain ER2738-1 is as follows.

Method of mosaicking the mutant lpdA gene into the chromosome of the transgenic strain ER2738-1: In this example 1, the method of mosaicking the mutant lpdA gene into the chromosome of the transgenic strain ER2738-1 is based on the previously published literature.的方法(Chiang et al,2012.Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites.Proc Biochem 47:2246-2254; Chiang et al,2008.Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli. Biotechnol Bioeng 101: 985-995).

First, a series of plastids with mutant lpdA gene were constructed by using the bacteriophage λ, the bacteriophage Φ80, the bacteriophage P21, the recombinase Int and Flp of the bacteriophage HK. The kanamycin-resistant gene with the attachment site and the FRT sequence at both ends. The aforementioned plastids are genetically recombined with the probacterial mosaic position on the chromosome by the action of the bacteriophage recombinase Int, and the aforementioned plastids are then embedded into the probacterial mosaic position on the chromosome of the transgenic strain ER2738-1 .

Screening method of the transgenic strain ER2738-1: In this example 1, the transgenic strain ER2738 was successfully inserted into the donor DNA that enhanced the expression of zwf gene, the donor DNA that enhanced the expression of aceEF gene, and the donor DNA that enhanced the expression of udhA gene. The screening method of -1 is to screen out the transgenic strain ER2738-1 on the solid LB medium containing kanamycin, and then assist the pTH19-CreCs to remove the mosaic in the gene of the transgenic strain ER2738-1 kan marker gene. The preparation of pTH19-CreCs of plastids is based on Chiang et al, 2012. Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites. Proc Biochem 47: 2246-2254.

In Example 1, the method for screening the transgenic strain ER2738-1 that successfully inserted the donor DNA of the mutant lpdA gene was to select the transgenic strain ER2738-1 on solid LB medium containing kanamycin. Then, Flp recombinase removes the kanamycin-resistant gene in the plastid containing the mutant lpdA gene and the replication origin of the plastid that accompanies the insertion.

Preparation method of donor DNA: In this example 1, the preparation method of donor DNA to enhance the expression of zwf gene is to amplify the upstream region and 5'end structural gene fragments of E. coli zwf gene individually, and then use PCR amplification Obtain a gene fragment containing the λ phage PR promoter and LE*-kan-RE* cassette (LE*-kan-RE* cassette contains the Kanamycin-resistant gene kan). The above-mentioned gene fragment is T4 The DNA ligase ligation combination produces donor DNA that enhances the expression of zwf gene. Here, the method of enhancing zwf gene expression refers to the method used in the previously published literature (M. Saini et al. 2016. Systematic engineering of the central metabolism in Escherichia coli for effective production of n-butanol. Biotechnol for Biofuels 9: 69).

In Example 1, the preparation method of the donor DNA for enhancing the expression of the aceEF gene is the same as the preparation method of the donor DNA for enhancing the expression of the zwf gene. Here, the method of enhancing aceEF gene expression refers to the method used in the previously published literature (M.Saini et al.2016.Systematic engineering of the central metabolism in Escherichia coli for effective production of n-butanol.Biotechnol for Biofuels 9: 69).

Comparison of growth status and phage yield between the infected transgenic strain ER2738-1 and the original strain ER2738: Firstly, the transgenic strain ER2738-1 was prepared according to the aforementioned process, and then the protozoan strain ER2738 and the transgenic strain ER2738-1 were respectively infected with M13 phage to obtain the infected protozoan strain ER2738 (M13) and the transgenic strain ER2738-1 (M13). ).

Next, take 50 μl of the bacterial solution of the native strain ER2738 (M13) and 50 μl of the bacterial solution of the transgenic strain ER2738-1 (M13), respectively inoculate 5 ml of M9-YM medium (containing glucose (2g/L)) for culture, Measure the cell density of the native strain ER2738 (M13) and the transgenic strain ER2738-1 (M13) every hour by measuring the native strain ER2738 (M13) and the transgenic strain ER2738-1 at the OD500 wavelength through a spectrophotometer. (M13) The absorbance value of the bacterial liquid, and at the end of 8 hours of incubation, sample the bacterial liquid from the original strain ER2738 (M13) and the transgenic strain ER2738-1 (M13), and calculate the original strain by the aforementioned virus plaque counting method The number of M13 phage produced by ER2738 (M13) and the transgenic strain ER2738-1 (M13).

The above experimental results are shown in Figure 2. Compared with the protobacterium ER2738, the growth of the protozoa strain ER2738 (M13) after infection with the M13 phage was significantly inhibited, and the amount of M13 phage produced by the protozoa strain ER2738 (M13) reached about 1.1 ×10 ¹¹ pfu/mL; In addition, the number of growth of the M13 phage-infected transgenic strain ER2738-1 (M13) was better than that of the M13 phage-infected proto-strain ER2738 (M13), but it was still slightly inferior to the uninfected ER2738-strain, and Strain ER2738-1 (M13) can produce M13 phage up to about 4.1×10 ¹¹ pfu/ml, and the output is increased by about 2.7 times. This result reveals that the pentose phosphate pathway and glycohydrolysis pathway of the transgenic strain ER2738-1 are enhanced as mentioned above, and the NADH production is increased, which can further increase the energy charge of the transgenic strain ER2738-1, thereby improving the resistance to M13. The growth status of host strains infected by phage and enhance the ability of host strains to produce M13 phage.

Example 2. Experiment of strengthening the electron transport chain of strain ER2738: In Example 2, the transgenic strain ER2738-2 that strengthens the electron transport chain path of the strain ER2738 was prepared to solve the problem that the ability of the host bacteria to produce M13 phage was inhibited.

The mechanism to solve the problem of inhibiting the ability of host bacteria to produce M13 phage by strengthening the electron transport chain of the host strain is as follows: As shown in Figure 1, the function of the cell electron transport chain can oxidize NADH in the cell to produce Proton motive force, adenosine triphosphate (ATPase) in the electron transport chain uses proton motive force to generate ATP. Therefore, by strengthening the electron transport chain path of the cell, the energy charge in the cell can also be increased.

The first way to strengthen the function of the cell electron transport chain is to increase the proton motility by increasing the number of protons produced by NADH oxidation in the cell electron transport chain. To increase the number of protons produced by NADH oxidation, you can strengthen NADH dehydrogenation. The performance of enzyme (NADH dehydrogenase ( ndh1 )) is achieved. NADH dehydrogenase participates in the reaction of the electron transport chain and has the function of proton pump. NADH dehydrogenase can increase the number of protons produced by NADH oxidation.

The second way to strengthen the function of the cell's electron transport chain is to allow more oxygen to participate in the electron transport chain. Oxygen acts as an electron receiver in the electron transport chain, that is, oxygen in the electron transport chain can contribute to the generation of protons. . Therefore, when the oxygen concentration in the cell is increased, the proton motility will also increase. It is currently known that the myoglobin and hemoglobin of eukaryotic cells have the function of carrying oxygen and increasing the oxygen concentration in the cell. In prokaryotes, the hemoglobin (VHb) structure of Vitreoscilla It is very similar to the myoglobin and heme structure of eukaryotic cells. Therefore, the hemoglobin of Vitreoscilla can also be combined with oxygen. When Vitreoscilla is in an oxygen-poor environment, the hemoglobin of Vitreoscilla can be used Help cells get from the environment Get oxygen. Therefore, by allowing the host strain to produce Vitreoscilla hemoglobin VHb, the oxygen concentration in the host strain can be increased, thereby strengthening the function of the electron transport chain in the host strain.

In this example 2, the transgenic strain ER2738-2 was enhanced to express the NADH dehydrogenase gene ndh1 and the transgenic strain ER2738-2 to obtain the hemoglobin gene vhb expressing Vitreoscilla, thereby enhancing the transgenic strain The energy charge of ER2738-2.

The preparation process of the transgenic strain ER2738-2 is as follows.

In Example 2, _{the donor DNA of the following bacteriophage PL} promoter was sent to the strain ER2738 for genetic recombination: the donor DNA to enhance the expression of the NADH dehydrogenase gene ndh1 gene of E. coli and the donor DNA containing the hemoglobin gene vhb .

In this example 2, the donor DNA that enhances the expression of the ndh1 gene was inserted into the chromosome of the transgenic strain ER2738-2 through the aforementioned DNA transformation method and host strain gene recombination method. The preparation system of the donor DNA of this example 2 refers to The preparation method of the donor DNA in Example 1 is cited in the literature. However, the method of embedding the hemoglobin gene vhb of Vitreoscilla is not using the aforementioned λ Red homologous recombination system. The method of inserting the donor DNA containing the hemoglobin gene vhb into the chromosome of the transgenic strain ER2738-2 is by constructing the chromosome of the transgenic strain ER2738-2. The pGB-TV2 pGB-TV2 of the hemoglobin gene vhb of the bacterium was transferred into the transgenic strain ER2738-2 by the aforementioned DNA transformation method for genetic recombination. The production of pGB-TV2 refers to the patent I305230 of the Republic of China.

The growth status and phage yield comparison experiment between the infected transgenic strain ER2738-2 and the original strain ER2738: In this example 2, the growth status and phage yield comparison experiment between the infected transgenic strain and the original strain in Example 1 are used In the experimental method, the growth status and phage yield of the infected transgenic strain ER2738-2 and the original strain ER2738 were compared.

The above-mentioned experimental results are shown in Figure 3. The growth status of the M13 phage-infected transgenic strain ER2738-2 (M13) is better than that of the M13 phage-infected protist strain ER2738 (M13), and is comparable to the uninfected protist strain ER2738 (see Figure 2 ), the M13 phage produced by the M13 phage infection transgenic strain ER2738-2 (M13) reached 4.5×10 ¹¹ pfu/mL, compared with the M13 phage output of the M13 phage infection of the protozoan strain ER2738 (M13), the M13 phage infection The production of M13 phage of the transgenic strain ER2738-2 (M13) increased approximately 3.1 times. This result reveals that the transgenic strain ER2738-2 can increase the production of ATP by strengthening the electron transport chain, further increase the energy charge of the transgenic strain ER2738-2, thereby improving the growth status of the host strains infected with M13 phage, and enhancing the host The ability of the strain to produce M13 phage.

Example 3. Experiment on the cell membrane structure of the modified strain ER2738: This example 3 solves the problem that the ability of the host bacteria to produce M13 phage is inhibited by preparing a transgenic strain ER2738-3 with modified cell membrane structure.

The mechanism to solve the problem of inhibiting the ability of host bacteria to produce M13 phage by modifying the cell membrane structure of the host strain is as follows: The secretion mechanism of M13 phage is through its pIV protein in the inner membrane of E. coli Holes are formed in the outer membrane of the bacteria, and the DNA of M13 phage is pulled to the vicinity of the holes by its pV protein. The DNA of M13 phage is assembled with the pVIII protein and PIII protein of M13 phage, and finally Escherichia coli is secreted. Extracellular. Based on the above-mentioned secretion mechanism of M13 phage, if the bacterial membrane of E. coli is destroyed, it will facilitate the secretion of M13 phage in E. coli into E. coli. In this example, the bacterial membrane of E. coli is destroyed by inhibiting the expression of Lpp lipoprotein of E. coli. Lpp lipoprotein is used to connect the outer bacterial membrane of E. coli and the peptidoglycan layer of the cell wall. When Lpp lipoprotein, it will affect the large intestine rod The connection between the bacterial outer membrane of the bacteria and the cell wall makes the structure of the bacterial outer membrane of E. coli relatively loose. Therefore, if the lpp gene that expresses Lpp lipoprotein in Escherichia coli is deleted, the expression of Lpp lipoprotein can be inhibited, and the cell membrane structure of Escherichia coli is destroyed, which is beneficial to the secretion of M13 phage.

In Example 3, the cell membrane structure of the transgenic strain ER2738-3 was modified by inhibiting the expression of the lpp gene.

The preparation process of the transgenic strain ER2738-3 is as follows.

In this example 3, the donor DNA (donor DNA from strain JW1667(△lpp::kan)(E.coli genetic stock center)) embedded with the kanamycin-resistant gene was inserted into the transgenic strain. In the lpp gene of ER2738-3, the lpp gene of the transgenic strain ER2738-3 was destroyed to obtain the transgenic strain ER2738-3. The donor DNA containing the kanamycin-resistant gene is inserted into the lpp gene of the transgenic strain ER2738-3 through the aforementioned DNA transformation method and host strain gene recombination method.

In this example 3, the lpp gene of the transgenic strain ER2738-3 was destroyed by inserting the kanamycin-resistant gene, but in other examples, other DNA fragments could still be inserted to destroy the transgenic strain ER2738- The lpp gene of 3 is not limited to Example 3.

In Example 3, the transgenic strain ER2738-3 that successfully destroyed the lpp gene contains the kanamycin resistance gene, so the transgenic strain ER2738-3 can be screened by referring to the screening method in Example 1.

The growth status and phage yield comparison experiment between the infected transgenic strain ER2738-3 and the original strain ER2738: In this example 3, the growth status and phage yield comparison experiment between the infected transgenic strain and the original strain in Example 1 were used In the experimental method, the growth status and phage yield of the infected transgenic strain ER2738-3 and the original strain ER2738 were compared.

The results of the experiment are shown in Figure 4. The growth status of the M13 phage infected transgenic strain ER2738-3 (M13) is comparable to that of the M13 phage infected prototypic strain ER2738 (M13), but not as good as the M13 phage infected transgenic strain ER2738-2 (M13). Growth status (as shown in Figure 3), but M13 phage infected with the transgenic strain ER2738-3 (M13) produced up to 5.9×10 ¹¹ pfu/mL, compared with M13 phage infected with the M13 of the proto-strain ER2738 (M13) Compared with the production of phage, the production of M13 phage infected with the transgenic strain ER2738-3 (M13) increased by approximately 4.4 times. This result reveals that the transgenic strain ER2738-3 can modify the structure of the cell membrane to make it a "loose" structure. The result of this modification of the cell membrane may reduce the energy charge state of the host strain (that is, the cell growth is inhibited), but it can improve The ability of host strains infected with phage to produce M13 phage.

Example 4. Global transcription machinery engineering experiment of strain ER2738: In this example 4, the transgenic strain ER2738-4 and the transgenic strain ER2738 with altered transcription function were prepared through global transcription machinery engineering. -5, to solve the problem that the host bacteria's ability to produce M13 phage is inhibited.

The principle of the overall transcription mechanism engineering to solve the problem that the host bacteria's ability to produce M13 phage is inhibited by changing the transcription of the host strain is as follows: The overall transcription mechanism engineering is a technology that can change the transcription performance of the chromosomal genes of the strain, more specifically In other words, it changes the ability of RNA polymerase (RNA polymerase) to recognize and activate various types of gene promoters during the transcription process, thereby inducing or inhibiting gene expression of specific proteins, allowing strains to exhibit different traits, such as: performance Outcomes the traits to resist harsh environments and the traits to increase metabolites.

In this example 4, in order to further improve the growth inhibition of the transgenic strain ER2738-3 caused by the deletion of the lpp gene in example 3, the RNA polymerase sigma factor D (RNA polymerase sigma factor D) in the ER2738-3 strain was changed. ) The function of the gene rpoD and the RNA polymerase sigma factor gene rpoS (RNA polymerase sigma factor S). The sigma factor is a promoter in E. coli that can regulate RNA polymerase to recognize different attributes. Among them, the rpoD gene participates in the regulation of the housekeeping gene of E. coli, and the housekeeping gene is related to the basic growth and metabolic activities of E. coli; The rpoS gene is involved in the regulation of the expression of heat shock genes, which are related to the growth and metabolism of Escherichia coli under adversity. That is, in this example 4, by inhibiting the normal growth and metabolic operation of the host strain, the host strain is inhibited from producing a large amount of phage shock protein (to combat the growth adversity of the host strain caused by the infection of M13 phage) to solve the host The strain produces a large amount of phage shock protein, which leads to the problem of poor energy status of the host strain.

In this example 4, by preparing a transgenic strain ER2738-4 with a mutation in the rpoD gene and a transgenic strain ER2738-5 with a mutation in the rpoS gene, the energy status of the host strain caused by the production of a large amount of phage shock protein was solved. Poor problem.

The preparation process of the transgenic strain ER2738-4 is as follows.

First, construct the pTH-rpoD plastid containing the rpoD gene. The RD-1 primer (gaagaattctcgccctgttccgcag) and the RD-2 primer (aagcaagcttaatcgtccaggaagctacg) were designed according to the rpoD gene of the E. coli strain, and the chromosome of the E. coli strain ER2738 was used as a template, using the RD-1 primer (contains the restriction enzyme EcoR I cut site) Perform PCR with the RD-2 primer (contains the restriction enzyme Hind III cutting site) to amplify a DNA fragment containing the rpoD gene. After cutting with restriction enzyme EcoR I and restriction enzyme Hind III to obtain PCR DNA fragment and plastid pTH18cr (National Institute of Genetics, Japan), use T4 cohesive enzyme to join the aforementioned cut DNA fragment and the cut plastid pTH18cr , To get pTH-rpoD.

Then use error-prone PCR (error-prone PCR) to amplify the sequence of pTH-rpoD (perform three consecutive wrong-pair PCR on pTH-rpoD) to obtain pTH-rpoD* containing the mutant rpoD gene. , The plastid pTH-rpoD* was then transferred into the transgenic strain ER2738-3 through the aforementioned DNA transformation method, thereby producing the transgenic strain ER2738-4.

The preparation process of the transgenic strain ER2738-5 is as follows.

First, construct pTH-rpoS containing rpoS gene, and design RS-1 primer (gaagaattccaccgttgctgttgcg) and RS-2 primer (aagcaagcttactcgcggaacagcgcttc) according to rpoS gene of E. coli strain, using E. coli strain ER2738 chromosome as template, using this RS-1 primer (gaagaattccaccgttgctgttgcg) PCR was carried out using primers (containing the restriction enzyme EcoR I cleavage site) and RS-2 primer (including the restriction enzyme Hind III cleavage site) to amplify a DNA fragment containing the rpoS gene. After cutting with restriction enzyme EcoR I and restriction enzyme Hind III to obtain PCR DNA fragment and plastid pTH18cr (National Institute of Genetics, Japan), use T4 cohesive enzyme to join the aforementioned cut DNA fragment and the cut plastid pTH18cr , To get pTH-rpoS.

Then use the error-prone PCR (error-prone PCR) method to amplify the sequence of the plastid pTH-rpoS (perform three consecutive mismatch PCRs on the pTH-rpoS of the plastid ) to obtain the pTH-rpoS* containing the mutant rpoS gene. , The plastid pTH-rpoS* was then transferred into the transgenic strain ER2738-3 through the aforementioned DNA transformation method, thereby producing the transgenic strain ER2738-5.

The error-prone PCR method used in Example 4 is as follows: In this example 4, the GeneMorph II Random Mutagenesis Kit produced by Agilent is used to perform mispairing PCR. The GeneMorph II Random Mutagenesis Kit uses the mutant DNA polymerase characteristics of Mutazyme II to improve DNA fragment amplification. The probability of error in order to achieve the effect of gene mutation. This set can control the mutation rate by adjusting the concentration of template DNA and the number of PCR cycles to facilitate related mutation experiments.

The growth status and phage yield comparison experiment of the infected transgenic strain ER2738-4, the transgenic strain ER2738-5 and the original strain ER2738: In this Example 4, the comparison of the infected transgenic strain and the original strain in Example 1 was used. The experimental method of the growth status and phage production comparison experiment was to carry out the growth status and phage production comparison experiment of the infected transgenic strain ER2738-4, the transgenic strain ER2738-5 and the original strain ER2738.

The experimental results are shown in Figure 5. The growth status of M13 phage infected transgenic strains ER2738-4 (M13) and ER2738-5 (M13) is better than that of M13 phage infected prototypic strain ER2738 (M13), which represents the through mutant type. The performance of rpoD and rpoS can promote the growth of M13 phage-infected transgenic strain ER2738-3, and is superior to the growth state of M13 phage-infected protozoan strain ER2738 (M13). In addition, the production of M13 phage from the transgenic strains ER2738-4 (M13) and ER2738-5 (M13) increased by M13 phage, and the yield of M13 phage from the transgenic strain ER2738-4 (M13) by M13 phage can reach 7.2×10 ¹¹ pfu/mL, the M13 phage produced by the M13 phage infection of the transgenic strain ER2738-5 (M13) can reach 10.1×10 ¹¹ pfu/mL, especially when the M13 phage infects the transgenic strain ER2738-5 (M13) and the M13 phage infects the native Compared with strain ER2738 (M13), the amount of phage produced by the former is about 10 times that of the latter. This result reveals that the M13 phage infection of the transgenic strains ER2738-4 (M13) and ER2738-5 (M13) can change the transcriptional performance of the host strain, thereby inhibiting the host strain’s production of a large number of phage shock proteins, which can effectively promote the strain’s energy charge. The gene expression of the status to solve the problem of the host strain's poor energy status due to the production of a large amount of phage shock protein.

The above-mentioned transgenic Escherichia coli can effectively increase the energy charge of the host strain by strengthening the sugar metabolism pathway of the host strain, strengthening the electron transport chain pathway of the host strain, inhibiting the lpp gene expression of the host strain, and inhibiting the transcription performance of the host strain. Improve the growth status of host strains infected with M13 phage and enhance the ability of host strains to produce M13 phage.

The present invention has been disclosed in a preferred embodiment above, but those skilled in the art should understand that the embodiment is only used to describe the present invention and should not be construed as limiting the scope of the present invention. It should be noted that all changes and substitutions equivalent to this embodiment should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be defined by the scope of the patent application.

Claims (2)

A method for preparing Escherichia coli for the production of M13 bacteriophage, which comprises the following steps: (a) providing Escherichia coli ER2738; (b) transfecting the mutant rpoS gene or the mutant rpoD gene into the Escherichia coli ER2738, and the mutant rpoS gene Or the mutant rpoD gene is prepared by using a mismatched PCR method; and (c) the mutant rpoS gene or the mutant rpoD gene is transformed into the Escherichia coli ER2738 in a plastid form, thereby changing the transcriptional effect of the Escherichia coli in vivo . A method for producing M13 phage, comprising the following steps: (a) providing Escherichia coli prepared by the method described in claim 1; and (b) infecting the Escherichia coli with M13 phage.

Images