Escherichia coli engineering bacteria and a method for preparing gene 3 knockout helper phage thereof

Abstract

This invention relates to an engineered *Escherichia coli* strain and a method for preparing gene 3 knockout helper phages. The genome of the engineered *E. coli* strain integrates a DNA module containing elements such as T7 Lysozyme, araC, ParaBAD, T7 RNA polymerase, PT7-lacO, and Ff gene 3. Through dual induction with L-arabinose and IPTG, tight regulation and high-level expression of the g3p protein can be achieved. Its property of strictly limiting leakage expression effectively reduces g3p protein degradation and misfolding. Using this strain to produce gene 3 knockout helper phages significantly improves the packaging efficiency and yield of the helper phages, reaching ≥ 8.5 × 10⁻⁶. 14 The pfu / L concentration is at least 600 times higher than existing technologies, significantly reducing production costs. This invention has significant application value in the field of phage display technology.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an engineered Escherichia coli strain and a method for preparing gene 3 knockout helper phages. Background Technology

[0002] Phage display technology, the first DNA-encoded screening technique, can screen for peptides or proteins encoded by genes with strong affinity for targets, and is therefore widely used in the development of emerging drug molecules such as antibodies and cyclic peptides. Initially, phage display involved directly fusing a library to the amino terminus of the mature protein of a few capsid proteins—the filamentous phage gene 3 protein (g3p), a process known as type 3 display. Later, a 3+3 display consisting of a phage particle and a helper phage was developed. The phage particle expresses the fusion protein of the library and g3p, while the helper phage expresses all phage proteins. Due to the unique defect of the replication initiation site in the helper phage, while the phage particle possesses a complete filamentous phage replication initiation site, the probability of phage proteins packaging phage particle single-stranded DNA is more than 10 times higher than packaging helper phage single-stranded DNA. 3+3 display has almost become the standard practice for phage display of large libraries such as antibodies and enzymes, because the smaller molecular weight of the phage particle gives it a significant advantage in terms of higher transformation efficiency during library construction.

[0003] However, unlike type 3 display, only a small fraction of phages from the 3+3 display can display their library proteins. In the 3+3 display, it seems that both the phage particle and the helper phage express g3p, and the library fusion g3p expressed by the phage particle should be roughly equal to the wild-type g3p. However, in reality, because wild-type g3p is easier to assemble, the ratio is usually less than 1:100. Since each phage displays 5 g3p proteins, this means that less than 5% of the phages effectively display their library proteins. Therefore, phage libraries from the 3+3 display are mostly undisplayed, which significantly reduces the effective library capacity and the copy number of each library sequence.

[0004] In 2001, Stefan Dübel invented a gene 3 knockout helper phage, Hyperphage (Rondot et al. 2001, Nature. Biotechnology.). This helper phage forces the production of progeny phages that use a library fusion with g3p on the phage particle, which can greatly improve the display titer of the phage particle system and significantly reduce the generation of useless, non-display phages. Similar helper phage designs have existed before and since, such as R408d3 (Rakonjac et al. 1997, Gene.), Ex-phage (Baek et al. 2002, Nucleic Acids Research. KR100458083B1), and Phaberge (Soltes et al. 2003, Journal of immunological methods, US20050130124A1), but their helper phage particle packaging yield and display titer are generally considered to be lower than Hyperphage (Soltes et al. 2007, Journal of Biotechnology). However, Hyperphage's yield is two orders of magnitude lower than that of the helper phage M13KO7 (without gene 3 deletion), at only 1.34 × 10⁻⁶. 12 The pfu / L bacterial culture significantly increases production and purification costs. Hyperphage is currently sold by Progen in Germany for €105 per vial (containing 2 x 10^6 bacteria). 12 PFU (particle-free phage) is very expensive. Antibody Design Labs in the US also sells a similar helper phage, CM13d3, but its price per unit phage titer is even higher. Therefore, there is a need for further improvement in existing gene 3 knockout helper phages and their production methods.

[0005] In recent years, several patents have disclosed the construction and application of similar gene 3 knockout helper phages, but the optimization of methods for producing such helper phages has not been mentioned. US12366009B2 discloses a method for the continuous evolution of target genes, enabling the continuous directed evolution of gene-encoded molecules that can be associated with proteins produced in host cells. By significantly accelerating laboratory evolution, PACE provides an effective solution to the difficult problem of directed evolution. It is also used for the continuous evolution of nucleic acids. The patent describes a class of helper phages that can express phage genes to assist in the packaging of phage particles into phage-like particles, but lack at least one phage gene and therefore cannot produce functional phages themselves. Such helper phages are often similar to gene 3 knockout helper phages, such as Hyperphage. Because such helper phages can exist continuously in the strain as plasmids, they do not need to be produced in engineered strains, thus avoiding the problem of low yield.

[0006] CN104428416B discloses a plasmid system that generates a phage displaying a Fab in *E. coli*, which then expresses and secretes a full-length IgG antibody containing this Fab in mammalian cells. The patent describes two helper phages that increase the display number of the displayed protein. These helper phages are similar to *Ex-phage* and *Phaberge*, with one or more amber stop codons added to M13KO7, allowing it to express the full-length g3p normally in amber-inhibited strains, thus achieving more efficient production. In strains without amber inhibition, these helper phages cannot express the full-length g3p, therefore, they are equivalent to gene 3 knockout helper phages when packaging helper phage particles.

[0007] CN113785058B discloses a phage display method for a polypeptide library containing non-classical amino acids, including library construction, inducing bacterial host translation of polypeptide sequences containing non-classical amino acids, and screening for peptides that bind to the desired target. The helper phage used in this method is the M13KO7 phage, which has a TAA mutation introduced at the K10 position of gene 3, similar to Ex-phage and Phaberge, so that the resulting progeny phages display only polypeptides containing non-classical amino acids. Summary of the Invention

[0008] The purpose of this invention is to overcome the problems of low yield and high production cost of existing gene 3 knockout helper phages, and to provide an engineered Escherichia coli strain and a method for preparing gene 3 knockout helper phages.

[0009] The *E. coli* engineered strain provided in this application is an *E. coli* strain that can controllably express the filamentous phage gene 3. Using this strain and plasmids containing publicly available gene 3 knockout helper phage sequences, gene 3 knockout helper phages can be prepared efficiently. Therefore, based on the engineered strain provided in this application, the production cost of gene 3 knockout helper phages is significantly reduced, allowing for wider application in new drug development.

[0010] The objective of this invention can be achieved through the following technical solutions: In a first aspect, this invention provides an engineered *Escherichia coli* bacterium that can controllably express the filamentous phage capsid protein g3p, named TT7O3in, and classified as *Escherichia coli*. Escherichia coli It was deposited on December 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37140. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0011] In one embodiment of the present invention, the engineered *Escherichia coli* strain (TT7O3in) is based on the *Escherichia coli* TG1 strain, whose genome integrates a foreign DNA module comprising the following elements: (a) a T7 lysozyme encoding gene; (b) an L-arabinose operon regulatory protein AraC encoding gene; (c) an arabinose promoter (ParaBAD); (d) a T7 RNA polymerase encoding gene with an amber mutation at position 114, the expression of which is controlled by the arabinose promoter (ParaBAD); (e) a ribosomal RNA terminator (rrnBterminator); (f) a T7 promoter / lactose operon complex element (PT7-lacO); (g) a filamentous phage gene 3 (Ffgene 3) encoding gene, the expression of which is controlled by the PT7-lacO element; and (h) a chloramphenicol resistance selection marker (CmR).

[0012] In one embodiment of the present invention, the engineered Escherichia coli (TT7O3in) also contains the gene encoding filamentous phage gene 6 (Ff gene 6). In this case, the engineered bacteria containing the gene encoding filamentous phage gene 6 (Ff gene 6) is called engineered Escherichia coli (TT7O36in).

[0013] The *E. coli* engineered strain TT7O3in constructed in this application achieves strictly inducible expression of the g3p protein through a multi-layered inhibition mechanism via its exogenous DNA module. Under conditions without inducers (IPTG and L-arabinose), normally expressed T7 lysozyme inhibits T7 RNA polymerase activity, while arabinose operon regulatory proteins inhibit the transcription of the T7 RNA polymerase gene by the arabinose promoter. Furthermore, the T7 RNA polymerase gene sequence contains a succinate mutation at position 114, which, even when read through in the TG1 host bacterium with a succinate inhibition phenotype, significantly reduces the expression level of the complete protein. Moreover, the lactose operon element downstream of the T7 promoter is inhibited by the host lacI protein. These multiple regulatory mechanisms work together to ensure that almost no fully functional T7 RNA polymerase is produced under induction-free conditions, thus preventing the initiation of downstream T7 promoter-driven filamentous phage g3p gene expression, making the g3p protein undetectable under normal conditions. When IPTG and L-arabinose are added simultaneously, the above inhibition is relieved, and T7 RNA polymerase is expressed efficiently, thereby driving high-level expression of g3p protein.

[0014] After the addition of the inducers L-arabinose and isopropyl-β-D-thiogalactoside (IPTG), the arabinose promoter is unrepressed, and T7 RNA polymerase is expressed in large quantities, overcoming the restriction of T7 lysozyme; the lactose operon following the T7 promoter is also unrestricted; therefore, filamentous phage gene 3 (which also contains filamentous phage gene 6 in the engineered E. coli TT7O36in) is transcribed and translated in large quantities. Although this gene has only one copy, its expression level of filamentous phage gene 3 can be close to 1 / 3 to 1 / 4 of the expression level of filamentous phage gene 3 (dozens of copies) in the M13KO7 host bacterium.

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned engineered Escherichia coli, wherein the exogenous DNA module is randomly integrated into the genome of Escherichia coli TG1 using a plasmid containing Tn5 transposase to obtain the engineered Escherichia coli.

[0016] In one embodiment of the present invention, a method for preparing an engineered Escherichia coli strain that can controllably overexpress the filamentous phage gene 3 is provided, comprising the following steps: (a) The exogenous DNA module to be integrated (containing the following elements: T7 lysozyme encoding gene, arabinose operon regulatory protein AraC encoding gene, arabinose promoter (ParaBAD), T7 RNA polymerase encoding gene, terminator, T7 promoter / lactose operon complex element, filamentous phage gene 3 encoding gene, filamentous phage gene 6 encoding gene (optional), and chloramphenicol resistance selection marker) is constructed between the two Tn5 ME sites of plasmid pBAM1 containing the Tn5 transposase gene by enzyme digestion ligation or Gibson assembly. (b) The plasmid pBAM1-Insert, which successfully constructs an introductory DNA module, was screened and amplified in strains with the pir+ genotype (such as S1030). (c) The plasmid pBAM1-Insert was electroporated into TG1 competent cells, and clones resistant to chloramphenicol were selected. (d) Verify the presence of exogenous DNA in the clone using colony PCR; (e) Select clones that do not contain plasmids by inoculating them on chloramphenicol and ampicillin plates; (f) The ability of the bacteria to induce the expression of g3p protein was verified by immunoblotting. The bacteria that showed no leakage of g3p expression without induction and showed high expression of g3p under induction were the engineered bacteria.

[0017] Furthermore, the integration of exogenous genes described in this application can also be implemented using the pKD-Tn5 plasmid constructed based on the pKD46 vector. pKD-Tn5 contains the gene encoding the thermosensitive replication factor rep101, its corresponding replication origin pSC101 ori, the ampicillin resistance gene AmpR, the λ phage Gam gene (which can inhibit bacterial RecBCD nuclease activity and prevent the degradation of transformed exogenous linear DNA) expressed under the control of the arabinose promoter, and the gene encoding the Tn5 transposase. The implementation steps are as follows: (a) pKD-Tn5 was transformed into Escherichia coli TG1 strain, single clones were selected, cultured at 30°C and induced with L-arabinose to prepare electrotransformation competent cells; (b) The linear exogenous DNA module to be integrated was directly transformed into the electrotransformation competent cells, plated, and the next day, single clones with chloramphenicol resistance were selected. (c) Selected single clones were incubated at 42°C for one day by shaking, and then streaked again on plates to select new single clones. Colony PCR was used to verify the presence of exogenous DNA in the clones. (d) Clones that lost the pKD-Tn5 plasmid were selected by inoculating chloramphenicol and ampicillin plates; (e) Verify its ability to induce the expression of g3p protein by immunoblotting. Those that do not leak expression without induction and express high levels of g3p under induction are the engineered bacteria.

[0018] Preferably, since the exogenous gene is large and transposition is difficult, the integration of the exogenous gene described in this application can also be performed by combining pBAM1-Insert and pKD-Tn5. Transforming pBAM1-Insert into the TG1 strain holding pKD-Tn5 has the advantage of significantly increasing the concentration of Tn5 transposase in the strain due to L-arabinose induction, and since pBAM1-Insert, as a circular DNA, is not degraded, it can improve the success rate of transposition integration.

[0019] In a third aspect, the present invention provides a method for preparing gene 3 knockout helper phages using the engineered Escherichia coli strain, the method comprising the following steps: S1: Cultivate the engineered Escherichia coli strain described above; S2: Infect the engineered bacteria with gene 3 knockout helper phage, or transform the engineered bacteria with a plasmid containing gene 3 knockout helper phage; S3: Pick out a single clone of the engineered bacteria that has been infected or transformed from a plate containing kanamycin, inoculate it into a liquid culture medium containing kanamycin, and culture it. S4: Introduce an inducer into the culture to induce high expression of g3p protein; S5: After culturing for a period of time, gene 3 knockout helper phage particles with infectious activity are packaged and amplified. S6: Harvest and purify the helper phage particles from the culture.

[0020] Preferably, the gene 3 knockout helper phage is selected from Hyperphage, HΔIL9, Hp6r2 or HDtD.

[0021] Preferably, in step S3, the culture is carried out at 37°C until the OD600 reaches 2-4; in step S4, the inducer is L-arabinose and IPTG; and in step S5, the culture is carried out at 30°C for another 10 to 16 hours.

[0022] In one embodiment of the present invention, a method for preparing a small-scale gene 3 knockout helper phage (Hyperphage) is provided, the preparation of which includes the following steps: (a) Infect Hyperphage into TT7O3in or TT7O36in strains, streak on a plate containing kanamycin, and incubate overnight at 37°C. The next day, select single clones and inoculate them into liquid LB medium containing kanamycin and chloramphenicol. Optionally, the liquid medium can be 2×YT or SOC. Note that transforming the Hyperphage plasmid will not yield clones that efficiently produce Hyperphage. In addition to infection, clones that efficiently produce Hyperphage can also be obtained by transforming ssDNA. Optionally, when producing HΔIL9, Hp6r2, and other phages, they can be introduced into TT7O3in or TT7O36in strains through transformation without reducing yield.

[0023] (b) The bacterial culture was cultured in a constant temperature shaker at 37°C until the OD600 was in the range of 2-4. Then, 1 mM IPTG and 2 mM L-arabinose were added to induce the expression of the filamentous phage gene 3.

[0024] (c) Transfer the bacterial culture to a 30°C constant temperature shaker and incubate overnight (8-16 hours) with shaking.

[0025] (d) Centrifuge the bacterial culture, discard the bacterial cells, and collect the supernatant containing Hyperphage. Add 1 / 4 volume or more of a precipitation reagent (20% polyethylene glycol 6000, 2.5 M sodium chloride) to the supernatant, freeze-centrifuge to precipitate Hyperphage, and resuspend Hyperphage in TBS. Repeat centrifugation to remove bacterial cells and purify Hyperphage. Alternatively, removal of bacterial cells and purification of Hyperphage can also be achieved by pressing the Hyperphage solution through a 0.45 μm aqueous filter membrane.

[0026] Furthermore, the cultivation in steps S3-S5 is carried out using a high-density fermentation process, ultimately achieving a helper phage yield of no less than 8.5 × 10⁻⁶. 14 pfu / L.

[0027] Furthermore, in one embodiment of the present invention, the specific steps for mass production of Hyperphage using a high-density fermentation process are as follows: (a) Hyperphage was infected into TT7O3in or TT7O36in strains, spread on agar plates containing kanamycin, and incubated overnight at 37°C. The next day, the mycelium was scraped off the agar plates and inoculated into 50 mL of liquid LB medium containing kanamycin and chloramphenicol, and activated by shaking at 37°C.

[0028] (b) Sterilize the fermenter containing the liquid culture medium by high-temperature steam. Inoculate the seed culture into the tank, then add kanamycin and chloramphenicol, and incubate until the OD600 reaches 20-30.

[0029] (c) Cool to 30°C, add IPTG and L-arabinose, and continue fermentation for 24 hours until dissolved oxygen is close to or exceeds 40%.

[0030] (d) Centrifuge the bacterial culture, discard the bacterial cells, and collect the supernatant containing Hyperphage. Add 1 / 4 volume or more of a precipitation reagent (20% polyethylene glycol 6000, 2.5 M sodium chloride) to the supernatant, freeze-centrifuge to precipitate Hyperphage, and resuspend Hyperphage in TBS. Repeat centrifugation to remove bacterial cells and cell debris, and purify Hyperphage. Optionally, cell removal and purification of Hyperphage can also be achieved by pressing the Hyperphage solution through a 0.45 μm aqueous filter membrane.

[0031] Preferably, when the produced gene 3 knockout helper phage is Hyperphage, a clone capable of efficiently producing Hyperphage can be obtained by infecting the strain with Hyperphage or by transforming the strain with Hyperphage ssDNA.

[0032] Compared with the prior art, the present invention has the following beneficial effects: 1. The engineered bacteria provided by this invention, because their exogenous DNA is integrated into the genome, will not mispackage or co-package plasmids into phages during the production of gene 3 knockout helper phages. If a strain expressing filamentous phage gene 3 with a plasmid is used, there is a probability of 1 / 1000-1 / 100 that the produced gene 3 knockout helper phages will co-package the plasmid within the strain.

[0033] 2. The gene expression module integrated into the engineered bacteria provided by this invention is highly controlled, effectively preventing the leakage expression of filamentous phage gene 3, thereby preventing the generation of misfolded and assembled g3p and g3p degradation products, and reducing the infectivity of the produced gene 3 knockout helper phage. Although the engineered bacteria have only one copy of filamentous phage gene 3, its g3p expression level is very high, which can effectively prevent insufficient g3p during the assembly of the produced gene 3 knockout helper phage, thus preventing reduced infectivity.

[0034] 3. According to previously published data, the yield of Hyperphage was only 1.34 × 10⁻⁶. 12pfu / L. According to the method provided by the present invention, particularly the clone obtained by infecting the strain TT7O3in with Hyperphage, the yield of Hyperphage can reach 2 × 10⁻⁶ pfu / L. 14 pfu / L. Furthermore, if other publicly available gene 3 knockout helper phages, such as Hp6r2, are used, the yield can be further increased to 6.6 × 10⁻⁶ pfu / L. 14 The pfu / L concentration of this helper phage is essentially equivalent to that of Hyperphage. If a high-density fermentation method is used, the yield can be increased to at least 8.5 × 10⁻⁶ pfu / L. 14 pfu / L, further reducing production costs. Attached Figure Description

[0035] Figure 1 A schematic diagram of the structure of the exogenous DNA module integrated by two engineered bacterial strains (TT7O3in and TT7O36in).

[0036] Figure 2 The process of strain preparation is shown in Figure 1. Figure 2 shows the agarose gel electrophoresis results of the pBAM1-Insert plasmid containing the gene module to be integrated. The plasmid in the left channel contains filamentous phage gene 3, and the plasmid in the right channel contains both filamentous phage gene 3 and gene 6. Figure 3 shows colony PCR of the six clones obtained during the construction of the TT7O3in strain, verified by agarose gel electrophoresis that they contain the gene module. Figure 4 shows the antibiotic resistance screening of the six clones obtained during the construction of the TT7O3in strain. The bacterial culture of the clones was dropped onto an ampicillin plate, and clones c3 and c6 showed unexpected ampicillin resistance, indicating that they contain the plasmid.

[0037] Figure 3 To test the expression of filamentous phage gene 3 in the strain under glucose inhibition (“Sup”, inhibition group), no inducer (“0”, control group), and the addition of L-arabinose and IPTG inducer (“Ind”, induction group); NC is the negative control, TG1 cells; PC is the positive control, TG1 cells carrying M13KO7; an equal amount of cells were used for polyacrylamide gel electrophoresis (SDS-PAGE). The lower half of the image shows the bacterial protein bands stained with Coomassie brilliant blue, serving as an internal control to confirm the cell quantity; the upper half of the image shows the chemiluminescent imaging of g3p by Western blotting using an antibody against filamentous phage g3p.

[0038] Figure 4The yield statistics of Hyperphage produced in small quantities by two strains (TT7O3in and TT7O36in) through infection and transformation are presented, and the yield is compared with that of Hyperphage produced by previous methods reported in the literature.

[0039] Figure 5 This study presents the yield statistics for small-scale production of HΔIL9 and Hp6r2 using two strains (TT7O3in and TT7O36in).

[0040] Figure 6 The titer of Hp6r2 produced by fermentation using the TT7O3in strain was determined by 8-fold purification and concentration of the phage before serial dilution.

[0041] Figure 7 This study describes the construction of the TT7O3in strain, its synthetic biology / metabolic engineering strategies, and related results. Detailed Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0043] Experimental materials: Escherichia coli TG1: purchased from LGC, Biosearch Technologies.

[0044] Vector: M13KO7 was purchased from New England Biolabs. Hyperphage and other gene 3 knockout helper phages were constructed based on M13KO7.

[0045] LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, add water to a final volume of 1 L. Solid LB medium: Add 15 g agar powder to the LB medium. Autoclave at 121℃ for 15 min.

[0046] 10% Glycerin: Add 100 mL of glycerin to water to 1 L, autoclave at 121°C, and refrigerate at 4°C.

[0047] Precipitation reagent: 20% polyethylene glycol 6000, 2.5 M sodium chloride, dissolved in water to 1 L.

[0048] Bacterial lysate: containing lysozyme, RNase, DNase, etc., purchased from Changzhou Boyi Biotechnology Co., Ltd.

[0049] Fermentation medium: 18.3 g dipotassium hydrogen phosphate trihydrate, 4 g potassium dihydrogen phosphate, 1.7 g citric acid, 1.2 g magnesium sulfate, 4 g tryptone, 3 g yeast extract, 1 g sodium chloride, 1 mL defoamer; Trace element solution: 0.24 g ferric chloride hexahydrate, 0.08 g cobalt chloride hexahydrate, 0.015 g copper sulfate pentahydrate, 0.03 g zinc sulfate heptahydrate, 0.03 g sodium molybdate dihydrate, 0.0075 g boric acid, 0.12 g manganese chloride, and 1 g calcium chloride dihydrate, dissolved in 100 mL of water, with 1 mL added per liter of fermentation medium; Feed culture medium: 20 g L-arabinose, 80 g glycerol, 1.3 g magnesium sulfate, 3 g peptone, 2.5 g yeast extract, add water to a final volume of 200 ml, autoclave at 121℃ for 15 min, cool and add chloramphenicol at a final concentration of 34 μg / mL and kanamycin at a final concentration of 50 μg / mL.

[0050] Example 1: Preparation and testing of engineered bacterial strain TT7O3in (optionally, or TT7O36in). Preparation of plasmid pBAM1-Insert Will as Figure 1 The exogenous DNA module to be integrated (containing the following elements: T7 lysozyme encoding gene, arabinose operon regulatory protein AraC encoding gene, arabinose promoter (ParaBAD), T7 RNA polymerase encoding gene, terminator (ter / rrnB), T7 promoter (PT7) / lactose operon complex element (lacO), filamentous phage gene 3 encoding gene (Ffgene3), filamentous phage gene 6 encoding gene (optional; if this gene is present, the resulting engineered bacterial strain is TT7O36in; if this gene is absent, the resulting engineered bacterial strain is TT7O3in), and chloramphenicol resistance selection marker (CmR)) is constructed between two Tn5 ME sites of plasmid pBAM1 containing the Tn5 transposase gene by enzyme digestion ligation or Gibson assembly. The plasmid pBAM1-Insert, which contains a foreign DNA module, was successfully constructed by screening and amplifying strains with the pir+ genotype (such as S1030). The agarose gel electrophoresis results of plasmid pBAM1-Insert are as follows: Figure 2 As shown in figure a, the plasmid construction was successful, as confirmed by the Sanger sequencing results.

[0051] 2. Preparation of TG1 electrocompetent bacterial culture: Streak the TG1 bacterial culture on antibiotic-free plates (solid LB medium) and incubate overnight at 37°C. Pick single colonies and incubate in 20 mL of antibiotic-free LB liquid medium at 37°C until the OD600 reaches approximately 0.3. Centrifuge at 8000 g for 1 min to collect the bacteria. Resuspend the bacteria in 10 mL of 10% glycerol solution on ice, centrifuge at 8000 g for 1 min, and discard the supernatant. Repeat this process to wash the bacteria three times, and finally resuspend the bacteria in 0.5 mL of 10% glycerol solution to obtain the TG1 electroporation competent bacterial culture, which should be kept on ice for later use. Alternatively, it can be flash-frozen in liquid nitrogen and stored at -80°C. 3. Electroporation of pBAM1-Insert: Take 100 μL of the above TG1 electroporation competent bacterial culture and mix it with 1 μg of pBAM1-Insert plasmid. Carefully add the mixture to a 1 mm electroporation cuvette and incubate on ice for 20 min. Place the cuvette in a MicroPulser Electroporator (Bio-Rad) and electroporate using the Ec1 program (1.8 kV, one pulse). Quickly add 1 mL of antibiotic-free SOC medium to the bacterial culture and incubate at 37°C for 30 min to recover. Centrifuge at 8000 g for 1 min, discard the supernatant, concentrate the bacterial culture to 200 μL, and spread it completely on a plate containing 34 μg / mL chloramphenicol. Incubate overnight at 37°C.

[0052] 4. Selecting clones: The following day, colonies grew on the plate. Several single colonies (six were selected in this example) were picked and inoculated into 2 mL of LB medium containing 34 μg / mL chloramphenicol (c1, c2, c3, c4, c5, c6). 1 μL of each colony was used as a template for colony PCR to test for the presence of the key genes T7 RNA Pol and Ff gene 3. Colonies that did not contain the genes to be integrated were excluded. Figure 2 b, c4, and c5 are bacterial suspensions that do not contain the gene to be integrated. A small amount of bacterial suspension was taken with a pipette tip and added to 100 μg / mL ampicillin and 34 μg / mL chloramphenicol plates, respectively. The plates were incubated overnight at 37°C. Clones that could not grow on the ampicillin plate were picked from the chloramphenicol plate, such as… Figure 2 c, c1, and c2 are engineered bacterial strains TT7O3in that do not contain plasmids and have been integrated with foreign DNA modules.

[0053] The engineered *E. coli* strain in this embodiment controls for high expression of the filamentous phage capsid protein g3p, named TT7O3in, and classified as *Escherichia coli*. Escherichia coliIt was deposited on December 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37140. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0054] 5. Testing for controlled expression: Three parallel single colonies of engineered bacteria (inhibition group Sup, control group 0, and induction group Ind) were inoculated together with the negative control NC (TG1) and the positive control PC (TG1 containing M13KO7) into liquid LB medium containing the corresponding antibiotics (TT7O3in engineered bacteria: containing 34 μg / mL chloramphenicol; NC: no resistance; PC: 50 μg / mL kanamycin). An additional 20 mM glucose was added to the engineered bacterial culture (inhibition group). The culture was shaken until the OD600 reached approximately 1. Then, 1 mM IPTG and 2 mM L-arabinose were added to the engineered bacterial culture (induction group), and the culture was shaken again. After 3 hours, the bacterial cultures of each group were collected, and their OD600 was measured. According to 1 OD600 = 1.5 × 10⁻⁶, the OD600 of each group was calculated. 9 The bacterial concentration was calculated using CFU / mL. An equal volume of bacterial cells (4.5 × 10⁻⁶) was taken from each group of bacterial solutions. 8 CFU was centrifuged, the supernatant was discarded, and the bacterial cells were resuspended in 100 μL of bacterial lysis buffer and lysed for 10 min. 25 μL of 5×SDS loading buffer was added to each group, mixed thoroughly, and denatured at 98°C for 10 min. Samples were then loaded for SDS-PAGE electrophoresis (150 V, 1 h). The upper half of the gel was removed, and the protein was transferred to a formaldehyde-activated PVDF membrane in transfer buffer (330 mA, 1 h). The lower half of the gel was stained with Coomassie Brilliant Blue solution for 1 h, and then destained by shaking with destaining solution for 3 h. The image was recorded as a reference. The PVDF membrane carrying the protein was blocked with 10 mL of 5% skim milk solution at room temperature for 1 h. Immediately afterwards, 1 μL of α-g3p antibody (purchased from Thermo Scientific) and 1 μL of HRP-crosslinked goat anti-rabbit antibody were added, and the membrane was incubated at room temperature with shaking for 2 h. The PVDF membrane was washed three times with TBS, and then an enhanced chemiluminescent substrate (purchased from Shanghai Sangon Biotech Co., Ltd.) was added to the PVDF membrane in a chemiluminescence developer for development. The results are as follows. Figure 3 As shown, the TT7O3in strain does not express phage g3p at all without the addition of an inducer or glucose inhibition; however, it can express phage g3p efficiently after the addition of an inducer.

[0055] Example 2: A method for preparing a small-scale gene 3 knockout helper phage using engineered bacterial strain TT7O3in (optionally, or TT7O36in).

[0056] 1. Seed preparation: Hyperphage was infecting the TT7O3in strain and streaked onto a 50 μg / mL kanamycin plate. It was incubated overnight at 37°C, and single clones were picked the following day. Note that transforming the Hyperphage plasmid with dsDNA will not yield clones producing Hyperphage efficiently. Alternatively, in addition to infection, clones producing Hyperphage efficiently can also be obtained by transforming ssDNA. For other high-yield gene 3-knockout helper phages, both infection and transformation can be used to prepare high-yield seed clones.

[0057] 2. Cultivation: Single colonies were picked from the plates and inoculated into liquid LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol (the liquid volume should not exceed 10% of the container volume). The medium was incubated at 37°C with shaking until the OD600 reached 2–4. Then, 1 mM IPTG and 2 mM L-arabinose were added for induction. The medium was then transferred to 30°C and incubated overnight with shaking (8–16 h).

[0058] 3. Harvesting and purification: Harvest the bacterial culture, centrifuge at 8000 g for 30 min to precipitate bacterial cells and cell debris, discard the bacterial cells, and collect the supernatant containing Hyperphage. Add 1 / 4 volume or more of precipitation reagent to the supernatant, mix well, and pre-cool on ice for 1 h. Centrifuge at 10000 g for 45 min at 4°C to precipitate Hyperphage, and discard the supernatant. Resuspend Hyperphage by adding a small amount of TBS. If turbidity is visible to the naked eye, centrifuge again at 8000 g for 30 min to precipitate bacterial cells and cell debris. Repeat the centrifugation and phage precipitation process to further purify Hyperphage. Optionally, the clarified phage solution can be filtered through a 0.45 μm aqueous filter membrane to remove residual bacterial cells and cell debris.

[0059] 4. Counting: The titer testing method is as follows: Take 8-10 wells in a 96-well plate and add 90 μL of mid-log TG1 bacterial suspension (OD600 ~ 1). Add 10 μL of phage solution or phage-containing bacterial supernatant to the bacterial suspension in the first well to dilute it 10-fold. Then, take 10 μL from the first well and add it to the second well, and so on, performing a 10-fold serial dilution. Incubate the plate at 37°C for 30 minutes to allow phage infection of TG1 bacteria. Take 5 µL of bacterial suspension from each serially diluted solution, from low to high concentration, and spot it onto the marked position on the kanamycin-containing plate. After the plate has dried, incubate it overnight at 37°C and count the bacteria the following day.

[0060] like Figure 4 As shown, the Hyperphage yield (1.34 × 10⁻⁶) was compared with that of the conventional method (Rondot et al. 2001, Nat. Biotech.). 12 Compared to pfu / L, TT7O3in-based infection clones can significantly increase Hyperphage yield.

[0061] Based on this example, HΔIL9 and Hp6r2 were prepared in small quantities using engineered bacterial strains TT7O3in and TT7O36in, following a process similar to that used in the production of Hyperphage. The yield statistics for small-scale production of HΔIL9 and Hp6r2 using the two strains (TT7O3in and TT7O36in) are as follows: Figure 5 As shown.

[0062] Example 3: A method for preparing gene 3 knockout helper phages in large quantities by high-density fermentation using engineered bacterial strain TT7O3in (optionally, or TT7O36in).

[0063] 1. Seed preparation: Hyperphage was infected into the TT7O3in strain and spread on a 50 μg / mL kanamycin plate. It was incubated overnight at 37°C. The next day, the mycelial mat was scraped from the plate and inoculated entirely into 50 mL of liquid LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol. The medium was then incubated at 37°C with shaking for activation. It should be noted that transforming the Hyperphage plasmid with dsDNA will not yield a clone producing Hyperphage efficiently. Alternatively, in addition to infection, a clone producing Hyperphage efficiently can also be obtained by transforming ssDNA. For other high-yield gene 3-knockout helper phages, both infection and transformation can be used to prepare seed clones producing Hyperphage efficiently.

[0064] 2. Cultivation: The fermenter containing the liquid culture medium is sterilized by high-temperature steam. Once the cooling water system has cooled to below 37°C, the seed culture is inoculated into the fermenter, followed by the addition of kanamycin and chloramphenicol at a final concentration of 50 μg / mL. The fermenter is initially set to 37°C, with aeration at 3-7 L / min and stirring at 300 rpm. Dissolved oxygen is maintained at approximately 30%, with the associated stirring speed set to 300-800 rpm. The pH is associated with the acid-base pump, set to 7.0 and maintained between 6.9 and 7.5. Fermentation continues until the OD600 reaches 20-30. The temperature is lowered to 30°C, and IPTG and L-arabinose are added at a final concentration of 1 mM. Fermentation continues for 10 hours until dissolved oxygen begins to increase. At this point, fed culture medium is added, and fermentation continues for another 24 hours until dissolved oxygen approaches or exceeds 40%.

[0065] 3. Harvesting and purification: Same as Example 2.

[0066] 4. Counting: Same as Example 2, results are shown below. Figure 6 .

[0067] Figure 7 This is a schematic diagram summarizing the composition, function, and production performance of the TT7O3in strain. The TT7O3in engineered strain of this application, through an optimized gene expression regulation system, coupled with optimized culture methods or sequence-optimized helper phages, significantly solves the proliferation limitation problem of gene 3 knockout helper phages during production. Furthermore, high-density fermentation further improves the production efficiency of gene 3 knockout helper phages, thereby further reducing production costs. In summary, this strain provides an efficient production platform for industrial applications relying on phage display technology, such as antibody drugs and gene editing enzymes.

[0068] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An engineered strain of *Escherichia coli*, characterized in that, The controlled high expression of filamentous phage capsid protein g3p, named TT7O3in, was classified as Escherichia coli. Escherichia coli It was deposited on December 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37140. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The engineered Escherichia coli strain according to claim 1, characterized in that, The engineered Escherichia coli strain is based on the Escherichia coli TG1 strain, whose genome integrates a foreign DNA module. The foreign DNA module contains the following elements: a T7 lysozyme encoding gene, an L-arabinose operon regulatory protein AraC encoding gene, an arabinose promoter, a T7 RNA polymerase encoding gene, a ribosomal RNA terminator, a T7 promoter / lactose operon complex element, a filamentous phage gene 3 encoding gene, and a chloramphenicol resistance selection marker.

3. The engineered Escherichia coli strain according to claim 2, characterized in that, The engineered Escherichia coli strain also contains the gene encoding filamentous phage gene 6. In this case, the engineered strain containing the gene encoding filamentous phage gene 6 is called engineered Escherichia coli strain TT7O36in.

4. An engineered Escherichia coli strain according to claim 2 or 3, characterized in that, Without an inducer, the g3p protein is not expressed; under induced conditions, the strain expresses the g3p protein efficiently.

5. The method for preparing engineered Escherichia coli as described in claim 1, characterized in that, Using a plasmid containing the Tn5 transposase, the exogenous DNA module was randomly integrated into the genome of Escherichia coli TG1.

6. A method for preparing gene 3 knockout helper phages, characterized in that, The method includes the following steps: S1: Cultivate engineered Escherichia coli as described in any one of claims 1-4; S2: Infect the engineered bacteria with gene 3 knockout helper phage, or transform the engineered bacteria with a plasmid containing gene 3 knockout helper phage; S3: Pick out a single clone of the engineered bacteria that has been infected or transformed from a plate containing kanamycin, inoculate it into a liquid culture medium containing kanamycin, and culture it. S4: Introduce an inducer into the culture to induce high expression of g3p protein; S5: After culturing for a certain period of time, gene 3 knockout helper phage particles with infectious activity are packaged and amplified. S6: Harvest and purify the helper phage particles from the culture.

7. The method for preparing gene 3 knockout helper phage according to claim 6, characterized in that, The gene 3 knockout helper phage is selected from Hyperphage, HΔIL9, Hp6r2 or HDtD.

8. The method for preparing gene 3 knockout helper phage according to claim 6, characterized in that, In step S3, the culture is carried out at 37°C until the OD600 reaches 2-4; in step S4, the inducing agents are L-arabinose and IPTG; in step S5, the culture is carried out at 30°C for another 10 to 16 hours.

9. The method for preparing gene 3 knockout helper phage according to claim 6, characterized in that, The culture in steps S3-S5 was carried out using a high-density fermentation process, resulting in a final auxiliary phage yield of no less than 8.5 × 10⁻⁶. 14 pfu / L.

10. A method for preparing gene 3 knockout helper phage according to any one of claims 6-9, characterized in that, When the produced gene 3 knockout helper phage is Hyperphage, a clone capable of efficiently producing Hyperphage can be obtained by infecting the strain with Hyperphage or by transforming the strain with Hyperphage ssDNA.

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