Temperature-controlled cleavage plasmid based on nut site deletion of pL promoter as well as construction method and application of temperature-controlled cleavage plasmid

Abstract

The invention discloses a temperature-controlled cleavage plasmid based on nut site deletion of a pL promoter as well as a construction method and application of the temperature-controlled cleavage plasmid. According to the invention, on the premise of keeping the lambda cI857-pL / pR temperature control regulation mode unchanged, the stability of the system under medium-high temperature culture conditions is improved; meanwhile, compared with other systems for realizing thermal stability through promoter mutation, the system disclosed by the invention effectively relieves the problem that the cracking efficiency is obviously reduced after temperature rise induction by carrying out directional deletion transformation on a downstream regulation sequence of the promoter, so that the cracking process is more sufficient and reliable, and particularly, host bacteria can still be efficiently cracked under the induction condition of 42 DEG C; the bacteria dissolving efficiency can reach 99.99% or above, and the problem that the thermal stability and the cracking efficiency are difficult to consider in the bacterial ghost preparation process is solved.

Description

Temperature-controlled lysis plasmids based on pL promoter nut site deletion, their construction methods, and applications. Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to temperature-controlled lysis plasmids based on the deletion of the nut site of the pL promoter, their construction methods, and applications. Background Technology

[0002] Bacterial ghosts (BGs) are a type of bacterial shell formed by removing the cytoplasmic contents of bacteria through bioengineering. Because they retain the original bacterial morphology and surface antigen structure intact during formation, BGs can effectively stimulate humoral, cellular, and mucosal immune responses in the host. Furthermore, they possess certain adjuvant properties, making them a promising novel vaccine platform and a delivery carrier for antigens, nucleic acids, and other bioactive substances.

[0003] Currently, the controlled expression of the PhiX174 lysis gene E from bacteriophage, constructed using a temperature-controlled expression vector based on the λ phage promoter pL / pR and its thermosensitive repressor protein cI857, is a commonly used technique in bacterial shell preparation. In this system, the expression of the lysis gene E is effectively inhibited when the culture temperature is below 30℃ (usually 28℃); when the temperature rises above 30℃ (usually 42℃), the lysis gene E is expressed due to the thermal inactivation of the thermosensitive repressor protein cI857. Its product can form a transmembrane channel on the cell membrane, promoting the expulsion of cytoplasmic contents, ultimately resulting in a structurally intact bacterial shell.

[0004] The aforementioned temperature-controlled lysis system avoids the use of expensive chemical inducers, achieving the induction of lysis gene E expression solely through temperature changes, and boasts advantages such as ease of operation. However, this system still has certain limitations in practical applications. On the one hand, the required 28–30℃ culture conditions before induction are not the optimal growth temperature for many pathogenic or engineered bacteria, hindering rapid cell growth and potentially affecting the stable maintenance of surface antigen structures in some bacteria. On the other hand, the rapid temperature increase from 28℃ to 42℃ during induction can easily trigger a heat shock response in bacteria, thereby inhibiting the lysis process mediated by lysis gene E, reducing lysis efficiency and the quality of molted cell formation. Furthermore, achieving significant and rapid temperature transitions in the culture system under large-scale fermentation production conditions is more challenging than under laboratory conditions.

[0005] To mitigate the adverse effects of the aforementioned temperature limitations, previous studies have attempted to modify the pR or pL promoters through random mutation screening and site-directed mutagenesis verification. This has enabled the temperature-controlled system to stably suppress the expression of the lysis gene E at 37°C and 38°C, with some mutation combinations even raising the inhibition temperature to 39°C and continuing to induce bacterial lysis normally beyond its maximum inhibition temperature. However, such modifications are often accompanied by a significant decrease in lysis efficiency after temperature induction, making it difficult to simultaneously ensure both the thermal stability and lysis efficiency of the temperature-controlled system during molting preparation. This indicates that there is still room for further optimization and improvement of existing techniques.

[0006] Existing temperature-controlled lysis systems based on λcI857-pR / pL often suffer from a significant decrease in lysis efficiency mediated by the lysis gene E after promoter mutation modification to increase the inhibition temperature, making it difficult to meet the requirements of balancing thermal stability and lysis efficiency in the preparation of bacterial molts. Summary of the Invention

[0007] Purpose of the invention: The purpose of this invention is to provide a temperature-controlled lysis plasmid based on the deletion of the nut site in the pL promoter.

[0008] A second objective of this invention is to provide a method for constructing a temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter.

[0009] A third objective of this invention is to provide a recombinant bacterium.

[0010] A fourth objective of this invention is to provide the application of the temperature-controlled lysis plasmid and the recombinant bacteria in constructing a temperature-controlled lysis system.

[0011] Technical solution: The present invention provides a temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter. The temperature-controlled lysis plasmid includes a gene encoding the temperature-sensitive repressor protein cI857, a gene encoding the λ phage promoter pR, a pL promoter with the nut region deleted, a lysis gene E, and a transcriptional regulatory gene located downstream of the promoter.

[0012] The temperature-controlled lysis plasmid is obtained by directionally deleting the base sequence of the nut region downstream of the pL promoter in plasmid pKF396ME, using plasmid pKF396ME as the backbone.

[0013] The base sequence of the nut region is shown in SEQ ID NO.1. Preferably, the sequence of the temperature-controlled lysis plasmid is shown in SEQ ID NO.4.

[0014] This invention also provides a method for constructing a temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter, comprising the following steps:

[0015] (1) Primer pairs were designed based on the left promoter pL sequence, and the target fragment was obtained by PCR amplification using plasmid pKF396ME as a template;

[0016] (2) The purified target fragment and the linearized pKF396ME plasmid are ligated to obtain the temperature-controlled lysis plasmid.

[0017] The primer pair sequences in step (1) are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0018] The PCR amplification conditions in step (1) are: 98℃ for 10 s, 58℃ for 15 s, 72℃ for 30 s, for 30 cycles.

[0019] In step (2), the molar ratio of the target fragment to the linearized pKF396ME plasmid is 3~10:1.

[0020] In step (2), the linearized pKF396ME plasmid is obtained by double digestion of the pKF396ME plasmid with Xba I and EcoRI respectively.

[0021] The present invention also provides a recombinant bacterium containing the temperature-controlled lysis plasmid.

[0022] The recombinant bacteria are obtained by introducing the temperature-controlled lysis plasmid into a host bacterium, including Escherichia coli.

[0023] The present invention also provides the application of the temperature-controlled lysis plasmid and the recombinant bacteria in constructing a temperature-controlled lysis system.

[0024] This invention optimizes the structure of the transcriptional regulatory region downstream of the promoter in a temperature-controlled expression system based on λ phage cI857-pL. Specifically, the temperature-controlled lysis system of this invention includes the thermosensitive repressor protein cI857, λ phage promoters pR and pL, the lysis gene E, and transcriptional regulatory sequences located downstream of the promoters. Without affecting the integrity of the core regulatory elements of the pL promoter and its basic characteristics of being regulated by the thermosensitive repressor protein cI857, the transcriptional regulatory environment between the promoter and the downstream lysis gene is altered by deleting the nut region of the pL promoter, including boxA and boxB and their adjacent sequences. This allows the lysis gene E to be stably repressed under medium-high temperature conditions, while still efficiently lysing the host bacteria under 42℃ induction conditions, solving the problem of balancing thermal stability and lysis efficiency in the aforementioned bacterial molting preparation process. Furthermore, this invention provides an improved method for constructing the pL promoter nut region deletion mutant. This method directly splices homologous sequences that are inversely complementary to the template sequence on both sides of the region to be deleted in the reverse primer, so that the nut region to be deleted is directly crossed during the amplification process. Combined with conventional forward primers, the target deletion mutant can be obtained in a single round of PCR amplification, thereby avoiding the problems of multiple rounds of amplification, excessively short intermediate fragments, and low separation and recovery efficiency in the traditional overlapping PCR method, and improving the reliability and reproducibility of mutant construction.

[0025] Beneficial effects: Compared with the original plasmids based on the λcI857-pL temperature-controlled lysis system (such as pKF396ME) and the improved system that increases the inhibition temperature through pR or pL promoter mutation but is accompanied by a decrease in lysis efficiency, the present invention has the following beneficial effects:

[0026] (1) This invention improves the stability of the system under medium- and high-temperature culture conditions while maintaining the temperature control mode of λcI857-pL / pR. Furthermore, compared with other systems that achieve thermal stability through promoter mutation, this invention effectively alleviates the problem of significantly reduced lysis efficiency after temperature induction by directionally deleting the downstream regulatory sequence of the promoter, making the lysis process more complete and reliable. The recombinant bacteria containing the temperature-controlled lysis plasmid can still effectively inhibit the lysis gene E at 37℃, and especially under high-temperature induction at 42℃, it can still efficiently lyse the host bacteria, with a lysis efficiency of over 99.99%. This solves the problem of balancing thermal stability and lysis efficiency in the preparation of bacterial molts, achieving unexpected technical effects. Scanning electron microscopy and transmission electron microscopy observations show that the bacterial molts obtained by this plasmid preparation are basically consistent with the undiluted bacteria in overall morphology, with fully released cytoplasmic contents and intact bacterial shell structure, meeting the requirements for structural integrity and safety of vaccine vectors and biological delivery vectors. The recombinant bacteria containing this temperature-controlled lysis plasmid exhibit high inhibition temperature, high induction efficiency, and stable lysis effect. This effectively reduces the growth restriction and heat shock problems caused by low-temperature culture and drastic temperature rise induction in traditional temperature-controlled systems, making it suitable for preparing sclerotia of various Gram-negative bacteria. Furthermore, the lysis gene E in this system can be replaced with other functional genes as needed, thereby expanding its application scope in controlled gene expression and bioengineering.

[0027] (2) This invention does not require the introduction of additional expensive chemical inducers, and can still achieve stable inhibition and reliable induction under medium and high temperature conditions. It avoids the large and rapid increase in temperature required for induction in the original system, which can reduce the adverse effects of heat shock reaction of the host bacteria on the lysis process. It also helps to simplify the control conditions of fermentation and induction processes, improve the operational controllability and process stability in large-scale production, and provide a more stable technical foundation for the large-scale preparation of bacterial molt vaccines and related delivery vectors.

[0028] (3) This invention reduces the size of the final temperature-controlled lysis plasmid to 4027 bp by directionally deleting the nut region sequence 108 bp downstream of the pL promoter. This is smaller than the various temperature-controlled lysis systems based on λcI857 / pR and modified with mutations reported to date. The reduction in plasmid size helps to reduce the replication burden on the host bacteria, improves the stability of the vector during construction, transformation, and maintenance, and provides greater design space for the introduction of subsequent functional modules or application expansion.

[0029] (4) The method for constructing mutant plasmids with deletion of the nut region of the pL promoter proposed in this invention can achieve the target deletion by using a single round of PCR amplification. Without affecting the integrity of the core regulatory element of the promoter, it avoids the problems of low efficiency and high failure rate caused by multiple rounds of overlapping PCR amplification and short fragment separation and recovery in traditional methods. It significantly improves the reliability and reproducibility of mutant construction and provides a feasible technical path for the targeted modification of similar promoter internal sequences or regulatory elements. Attached Figure Description

[0030] Figure 1 shows the physical map of plasmid pKF396DL; cI857: thermosensitive repressor protein cI857; pR: lambda phage right-handed promoter; rrnbT1T2: ribosomal RNA operon T1T2 terminator; pLdn: left-handed promoter nut site deletion mutant; E: phage phiX174 lysis gene E; Ori: replication initiation region; Cat: chloramphenicol acetyltransferase gene.

[0031] Figure 2 shows the electrophoresis diagram of the PCR amplification product of the left promoter nut site deletion mutant pLdn; M: DNA Marker DL2000; 1-4: pLdn;

[0032] Figure 3 shows the sequence alignment results of the left-hand promoter pL of λ phage and the mutant pLdn with the nut site missing.

[0033] Figure 4 shows the lysis curve of Escherichia coli DH5α (pKF396DL);

[0034] Figure 5. Induction and inactivation of Escherichia coli DH5α (pKF396DL) at different temperatures;

[0035] Figure 6. Scanning electron microscopy (A, B) and transmission electron microscopy (C, D) observations of unlyslaught Escherichia coli DH5α and bacterial slough. Detailed Implementation

[0036] In the following embodiments, various processes and methods not described in detail are all conventional methods known in the art. Furthermore, the terminology used in this invention, unless otherwise stated, generally has the meanings commonly understood by those skilled in the art.

[0037] Experimental materials in this embodiment of the invention:

[0038] 1. Strains and plasmids

[0039] Escherichia coli DH5α chemocompetent cells were purchased from Nanjing Novizan Biotechnology Co., Ltd. (product number: C502-02). Plasmid pKF396ME was constructed and preserved in our laboratory (plasmid pKF396ME is derived from the article Fu LX, Gong JS, Gao B, Ji DJ, Han XG, Zeng LB. Controlled Expression of Lysis Gene E by a Mutant of the Promoter pL of the Thermo-inducible lambdacI857-pL System[J]. Journal of Applied Microbiology, 2021, 130: 2008-2017).

[0040] 2. Main reagents

[0041] Restriction endonucleases Xba I (Code No. 1634), EcoRI (Code No. 1611), T4 DNA ligase (Code No. 2011A), PrimeSTAR HS (Premix) (Code No. R040A), Premix Taq (Code No. RR901A), and DL2000 DNA Marker (Code No. 3427A) were all purchased from Takara Bio Inc. (Dalian). The Gel / PCR DNA Fragments Extraction Kit (Cat No. DF100) and Presto MiniPlasmid Kit (Cat No. PDH100) were Geneaid products. Primers were synthesized by Invitrogen (Shanghai) Trading Co., Ltd., a subsidiary of Thermo Fisher Scientific.

[0042] Example 1: Construction of the pKF396DL plasmid with deletion of the nut region of the pL promoter

[0043] Based on the known left-hand promoter pL sequence (based on the base sequence of the 665-727 region of the Lambda phage left-hand promoter nut deletion mutant in SEQ ID NO.4, combined with the upstream regulatory region and downstream sequence design), a pair of primers pLdn-F(5'-CTAC) containing Xba I and EcoRI restriction sites (underlined) and protective bases were designed. TCTAGA ACTAGTTCACCTACCAAAC-3') and pLdn-R(5'-CG GAATTCCTCCTTAATTTTTCCTGCTGATGTGCTCAGTA-3'), where the 5' end 19 nt sequence GAATTCCTCCTTAATTTTT and the 3' end 19 nt sequence CCTGCTGATGTGCTCAGTA both correspond to the original pL promoter sequence, but the two are 108 bp apart on the template. This gap region is the nut fragment containing boxA / boxB to be deleted.

[0044] Using plasmid pKF396ME as a template, the mutant left-handed promoter pLdn was amplified. The reaction volume was 50 µL, including 1 µL each of forward and reverse primers and template, 25 µL of PrimerStar HS Premix, and 22 µL of sterile double-distilled water. The PCR amplification conditions were: 98 °C for 10 s, 58 °C for 15 s, and 72 °C for 30 s, for 30 cycles. After the reaction, the PCR products were stained with bromophenol blue and electrophoresed on a 1.0% low-melting-point agarose gel. The target fragment was excised using a UV analyzer and recovered using a gel / PCR DNA Fragments Kit.

[0045] The recovered target gene fragment and pKF396ME plasmid were double-digested with Xba I and EcoRI, respectively. After purification, the target fragment was ligated into a plasmid vector digested with the same enzymes using T4 DNA ligase. The molar ratio of insert fragment to vector fragment in the ligation system was approximately 5:1. After gently mixing all components, ligation was performed overnight at 16°C. The next day, 10 µL of the ligation product was heat-shocked and transformed into *E. coli* DH5α competent cells. The transformed cells were then evenly spread on LB agar plates containing chloramphenicol (35 μg / mL) and incubated overnight at 28°C. Single colonies were picked for sequencing verification. The recombinant plasmid with the correct sequence was named pKF396DL, and its map is shown in Figure 1.

[0046] Using plasmid pKF396ME as a template, primer pair pLdn-F / pLdn-R successfully amplified the expected target fragment of approximately 173 bp (including restriction endonuclease and protective base sequences) (Figure 2). This fragment was recovered, digested, and cloned into plasmid pKF396ME, which had undergone the same digestion. Sequencing confirmed the successful construction of plasmid pKF396DL. Further sequence alignment showed that, compared to the original left-handed promoter pL, the modified pLdn fragment successfully deleted boxA and boxB elements at the nut site, resulting in a total deletion of 108 bp. Simultaneously, the key regulatory regions of the original pL promoter, including operon sequences OL1 to OL3, the transcription start site, and the ribosome binding site SD sequence, were completely preserved, as shown in Figure 3. The structure of pKF396DL is shown in Table 1, and its sequence is shown in SEQ ID NO.4.

[0047] Table 1. Vector-related information for plasmid pKF396DL

[0048] Example 2: Thermostability and Lysis Kinetics of Escherichia coli DH5α (pKF396DL)

[0049] Escherichia coli DH5α containing plasmid pKF396DL was streaked onto LB agar plates containing 35 μg / ml chloramphenicol and then incubated at 36℃, 37℃, 38℃, 39℃, and 42℃, respectively. Visible colonies of E. coli DH5α (pKF396DL) formed on the streaked LB agar plates at 36℃ and 37℃ under static incubation conditions, but no colonies were observed at 38℃, 39℃, and 42℃.

[0050] Single colonies of *E. coli* DH5α (pKF396DL) from streaked culture were inoculated into 100 ml of LB broth containing chloramphenicol (35 μg / ml) and cultured with shaking at 28°C. When the OD600 of the culture reached 0.3–0.4, the culture was further cultured at the original temperature (28°C) and at different temperatures (36°C, 38°C, 40°C, and 42°C). Samples were taken at regular intervals to measure the OD600 of the culture to monitor bacterial growth. At 0 h, 2 h, and 4 h after induction, 100 μL of bacterial culture was serially diluted 10-fold with sterile PBS and plated on LB agar plates, with three replicates for each dilution. After overnight incubation, viable cell counts were performed, and the lysis efficiency mediated by the lysis gene E was calculated. The calculation formula was: Lysis rate = (1 - CFU after induction / CFU before induction) × 100%.

[0051] As shown in Figure 4, the growth curves of *E. coli* DH5α (pKF396DL) under LB liquid medium culture conditions showed significant differences at different induction temperatures. At 28℃ and 36℃, the OD600 of the bacterial culture continuously increased over time after induction without a significant decline phase. Growth was faster at 36℃ in the first 2 hours, after which the growth rate and final OD600 value at 28℃ both exceeded those at 36℃. At 38℃, OD600 reached its peak at 120 min after induction, then declined and gradually stabilized. When the induction temperature increased to 40℃ and 42℃, the trend of OD600 changed significantly. OD600 rose briefly in the initial stage of induction before rapidly declining, with the decline at 42℃ being more rapid and dramatic, and the onset of the decline being relatively earlier.

[0052] Figure 5 shows the results of viable cell counts on plates, which reflects the effect of the lysis gene E and the thermostability of the system. At 28℃, the viable cell count continued to increase, increasing approximately 3.33 times after 4 hours compared to 0 hours. At 36℃, the viable cell count remained relatively stable, with the counts at 2 hours and 4 hours similar to the initial values. When the temperature rose to 38℃, the viable cell count showed a continuous downward trend, with lysis rates of 92.16% and 99.79% at 2 hours and 4 hours, respectively. When the induction temperature reached 40℃ and 42℃, the viable cell count decreased sharply. At 40℃, the viable cell count decreased by approximately 3-4 orders of magnitude compared to before induction, while at 42℃, the viable cell count decreased by approximately 4-5 orders of magnitude.

[0053] In summary, the Escherichia coli DH5α (pKF396DL) constructed in this invention can remain stable at 36-37℃, and can still be normally induced to lyse the host bacteria at 42℃. At the same time, it has a smaller volume, a higher lysis rate of Escherichia coli than other similar systems modified with temperature sensitivity, and can also reduce the heat shock response of the host bacteria.

[0054] Example 3

[0055] Following the same method as in Example 2, *Escherichia coli* DH5α (pKF396DL) was induced and cultured at 42°C. After 2 hours of induction, bacterial molts were collected and resuspended in 2.5% glutaraldehyde to adjust the bacterial concentration to 1×10⁻⁶. 8 CFU·mL -1The samples were fixed at 4°C for 2 hours. After fixation, the samples were washed three times with 0.1 mol / L phosphate-buffered saline (PBS) (10 minutes each time), and then post-fixed with 1% osmium tetroxide solution at room temperature in the dark for 1.5 hours. Subsequently, dehydration was performed using a gradient of ethanol (30%, 50%, 70%, 80%, 95%, and 100% v / v) and anhydrous isovalerate, 20 minutes per step. After final dehydration, critical point drying was performed using liquid carbon dioxide, and a gold-palladium alloy was sputtered on before scanning. Finally, the samples were observed and photographed under a scanning electron microscope.

[0056] The sample preparation process for transmission electron microscopy is the same as described above, but after gradient dehydration with ethanol, the sample is replaced twice with pure acetone (15 minutes each time). Then, the sample is placed in a mixture of acetone and resin (SPI-Pon 812R Embedding Kit, catalog number 02660R-AB) at a volume ratio of 3:1 and 1:1 for resin infiltration. The samples are treated at 37°C for 1 hour and 8-12 hours (overnight), respectively. Finally, the samples are immersed in pure resin. The resin-embedded sample blocks are polymerized at 60°C for 48 hours. After sectioning, the samples are stained with uranium acetate and lead citrate and are ready for observation.

[0057] Morphological observation of the surface and internal structure of *E. coli* DH5α vesicles was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM revealed that the external morphology of the vesicle cells was similar to that of unlyslaughtered bacteria, except for surface wrinkles and transmembrane lysis channels in the middle or at the poles of the cells (Fig. 6A, Fig. 6B). Further TEM revealed that the cytoplasmic contents of the vesicles were completely released, leaving only empty bacterial shells, while unlyslaughtered bacteria were full-bodied and contained intact cytoplasmic contents (Fig. 6C, Fig. 6D).

Claims

1. A temperature-controlled lysis plasmid based on the deletion of the nut site in the pL promoter, characterized in that, The temperature-controlled lysis plasmid includes a gene encoding the temperature-sensitive repressor protein cI857, a gene encoding the λ phage promoter pR, a pL promoter lacking the nut region, a lysis gene E, and a transcriptional regulatory gene located downstream of the promoter.

2. The temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter according to claim 1, characterized in that, The temperature-controlled lysis plasmid is obtained by directionally deleting the base sequence of the nut region downstream of the pL promoter in plasmid pKF396ME, using plasmid pKF396ME as the backbone.

3. The temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter according to claim 1, characterized in that, The base sequence of the nut region is shown in SEQ ID NO.

1. Preferably, the sequence of the temperature-controlled lysis plasmid is shown in SEQ ID NO.

4.

4. The method for constructing a temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter according to any one of claims 1 to 3, characterized in that, The steps include: (1) designing primer pairs based on the left promoter pL sequence and using plasmid pKF396ME as a template for PCR amplification to obtain the target fragment; (2) ligating the target fragment and linearized pKF396ME plasmid to obtain the temperature-controlled lysis plasmid.

5. The method for constructing a temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter according to claim 4, characterized in that, The primer pair sequences in step (1) are shown in SEQ ID NO.2 and SEQ ID NO.

3.

6. The method for constructing a temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter according to claim 4, characterized in that, The PCR amplification conditions in step (1) are: 98℃ for 10 s, 58℃ for 15 s, 72℃ for 30 s, for 30 cycles.

7. The method for constructing a temperature-controlled lysis plasmid based on the deletion of the nut site of the pL promoter according to claim 4, characterized in that, In step (2), the molar ratio of the target fragment to the linearized pKF396ME plasmid is 5:

1.

8. A recombinant bacterium, characterized in that, It contains the temperature-controlled lysis plasmid as described in any one of claims 1 to 3.

9. The recombinant bacteria according to claim 8, characterized in that, The recombinant bacteria are obtained by introducing the temperature-controlled lysis plasmid into a host bacterium, including Escherichia coli.

10. The application of the temperature-controlled lysis plasmid according to any one of claims 1 to 3 and the recombinant bacteria according to claim 8 or 9 in constructing a temperature-controlled lysis system.

Images