CN116926099A - Preparation method and application of physical stimulus control capable of generating bacterial cellulose and bacterial lysis and release of intracellular substances - Google Patents

Abstract

The present invention provides a preparation method and application for physical stimulation to control the lysis of engineered bacteria that can produce bacterial cellulose. Specifically, a plasmid for physically controlling bacterial lysis is disclosed. The plasmid has a physically activated promoter and is regulated by the physically activated promoter. The amplified gene encoding the cleavage protein, the gene encoding the cleavage protein also has a ribosome binding site upstream, and the ribosome binding site (RBS) has the sequence described in SEQ ID NO. 3, or SEQ ID NO. .3 has a sequence with 1, 2, 3 or 4 base mutations; the present invention also discloses an engineered bacterium that can produce bacterial cellulose containing the above plasmid. The present invention realizes self-clearance and release of intracellular substances by constructing a lysis system in the host bacteria, with controllable conditions and high accuracy. It achieves precise regulation of the lysis of the engineered bacteria by adjusting the RBS site of the lysis protein.

Description

A physical stimulus controls bacterial lysis and intracellular material release that generates bacterial cellulose Preparation methods and applications of radioactive

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a blue light-controlled Acetobacter xylinum lysis death and intracellular substance release system that can generate cellulose membranes.

Background technique

Bacteria grow quickly and are easy to cultivate on a large scale, so they have long been used by researchers to produce various drugs. With the development of synthetic biology, the use of engineered bacteria as drug delivery systems has unparalleled advantages over traditional drug delivery methods ^[1] . For example, bacteria can protect the activity of drugs and increase their half-life; more importantly, bacteria can deliver drugs to parts of the body that are difficult to reach by injection or oral administration.

Compared to other bacteria, microorganisms such as Acetobacter xylin can produce a strong and ultra-pure form of natural bacterial cellulose (BC). BC has good biocompatibility, can provide the best three-dimensional matrix for cell attachment, is non-toxic to a variety of cells, and can provide flexibility, high water retention capacity and gas exchange ^[2][3] . Although BC membranes have been widely used in food, medical and other fields, there have been very few modifications to the Acetobacter xylinum strain itself, making its function relatively single. In recent years, some researchers have tried to use Acetobacter xylinum to express some foreign proteins, but the problem of controlled release of intracellularly expressed proteins and other substances has not yet been solved ^[4] .

[1]Kang M,Choe D,Kim K,et al.Synthetic Biology Approaches in TheDevelopment of Engineered Therapeutic Microbes[J].Int J Mol Sci,2020,21(22):

[2] Picheth GF, Pirich CL, Sierakowski MR, et al. Bacterial cellulose inbiomedical applications: A review [J]. Int. J. Biol. Macromol, 2017, 104 (Pt A): 97-106.

[3]Barja F.Bacterial nanocellulose production and biomedical applications[J].J Biomed Res,2021,35(4):310-317.

[4]Florea M,Hagemann H,Santosa G,et al.Engineering control ofbacterial cellulose production using a genetic toolkit and a new cellulose-producing strain[J].Proc.Natl.Acad.Sci.U.S.A,2016,113(24 ):E3431-3440.

Contents of the invention

To solve the above problems, the present invention constructs a physically activated lysis system to control the self-clearance of bacteria that can generate bacterial cellulose membranes and the release of intracellular substances. The present invention uses synthetic biology methods to construct a lysis system regulated by physical stimulation in bacteria that can produce bacterial cellulose to remotely control the lysis of bacteria that can produce bacterial cellulose and the release of intracellular substances. Engineered bacteria that can produce bacterial cellulose can grow normally and produce BC membranes under dark conditions, but when physical stimulation is used, the bacteria that can produce bacterial cellulose lyse and release intracellular substances.

One aspect of the present invention provides a plasmid for physically controlling bacterial lysis. The plasmid has a physically activated promoter and a coding gene for a cleavage protein that is amplified under the control of the physically activated promoter. The upstream coding gene for the lysis protein also has Ribosome binding site, the ribosome binding site (RBS) has a sequence as described in SEQ ID NO. 3.

Further, the physically activated promoter is a promoter that can be initiated due to changes in light, temperature, pressure, and osmotic pressure.

Further, the light is preferably blue light.

Further, the physically activated promoter is selected from the blue light promoter pDawn, preferably, its sequence is shown in SEQ ID NO.1.

Further, the lytic protein is selected from proteins that can cause bacterial lysis, preferably lytic protein E of phage φ174, LKD16 phage lytic protein, and lambda phage lytic protein.

Further, the coding sequence of the lytic protein E of the phage φ174 is shown in SEQ ID NO. 2.

Further, the plasmid is selected from any plasmid that can be replicated in bacteria that can produce bacterial cellulose, for example, pSEVA331 is used as the vector plasmid.

Another aspect of the present invention provides an engineering bacterium capable of producing bacterial cellulose membrane (BC). The engineered bacterium capable of producing bacterial cellulose membrane has the above-mentioned plasmid for physical control lysis of the present invention and can be activated for lysis by physical stimulation.

Further, the host bacteria of the bacterial cellulose membrane engineering bacteria are selected from Acetobacter xylinum, Acetobacter pasteurianum, Acetobacter xylinum, Gluconacetobacter henselae, Acetobacter aceti, Acetobacter acetogenes, At least one of Aerobacter, Rhizobium, Achromobacter, Agrobacterium, Pseudomonas, Alcaligenes, Sarcina, and Kinectobacter.

Furthermore, the genome or plasmid of the engineered bacteria that can produce bacterial cellulose membranes can express genes encoding exogenous active substances.

Further, the active substance is selected from proteins, RNA, and polypeptides.

Another aspect of the present invention provides a method for constructing engineering bacteria that can generate bacterial cellulose membranes. The construction method includes:

S11) Construct a plasmid for physically controlling bacterial lysis. The plasmid has a physically activated promoter and a coding gene for a lysis protein that is amplified under the control of the physical activation promoter. The coding gene for the lysis protein also has a ribosome binding site upstream. , the ribosome binding site has the sequence described in SEQ ID NO.3;

S12) Transfer the plasmid for physically controlling bacterial lysis into wild-type bacteria that can produce bacterial cellulose membranes to obtain bacterial cellulose membrane-producing engineering bacteria that can control lysis under physical conditions.

Another aspect of the present invention provides the use of the plasmid for physically controlled lysis of the present invention in preparing bacteria that can be lysed by physical stimulation.

Yet another aspect of the present invention provides a method for regulating bacterial lysis and releasing intracellular substances produced by bacteria. The method includes:

S01) Construct the above-mentioned engineering bacteria that can produce bacterial cellulose membranes;

S02) Initiate physical stimulation to activate the lysis of the above engineered bacteria that can generate bacterial cellulose membranes.

Yet another aspect of the present invention provides a method for constructing engineered bacteria that can be induced to lyse by physical stimulation. The method includes the following steps:

S1) Select the corresponding promoter according to the type of physical stimulation, and select the lytic protein according to the type of bacteria;

S2) Use the random primer method to construct a mixed plasmid connection solution. The mixed plasmid connection solution contains the promoter selected in step S1), a series of different ribosome binding sites designed and obtained by the random primer method, and a series of different ribosome binding sites designed in step S1. ) The coding gene sequence of the selected cleavage protein;

S3) Transfer the mixed plasmid ligation solution obtained in step S2) into E. coli to obtain the E. coli engineering strain to be screened;

S4) Cultivate the E. coli engineered bacteria to be screened under the physical stimulation described in step S1) and under conditions other than the physical stimulation. The screening can obtain the results that can grow normally under the non-physical stimulation conditions and can grow normally under the physical stimulation conditions. E. coli strains that were completely lysed under the above physical stimulation conditions;

S5) Extract the corresponding recombinant plasmid from the E. coli engineering strain obtained by screening in step S4) and sequence it to obtain its corresponding ribosome binding site sequence, and introduce the recombinant plasmid into the wild-type bacteria described in step S1). , to obtain engineered bacteria that can be induced to lyse by physical stimulation.

Further, the bacteria are bacteria that can produce cellulose membranes that can produce exogenous proteins or target components.

Further, the physical stimulus is changes in light, temperature, pressure, and osmotic pressure.

beneficial effects

1. The present invention provides a way to control the lysis of engineered bacteria using a non-invasive induction method, which avoids the invasion of chemical inducers and the problem of diffusion in bacterial cellulose membranes. At the same time, this control method is not limited by time and space. .

2. The present invention uses a special method to solve the problem of leakage of lytic protein expression, resulting in the engineering bacteria not being lysed due to leakage of lytic protein expression without physical stimulation, and thus unable to obtain sufficient cellulose membrane or desired protein, or not. The effect of lysing the host bacteria is not achieved at the desired time. At the same time, the present invention also solves the problem that the lytic protein cannot reach the minimum threshold level of lysis after being activated by physical stimulation, and the host bacteria cannot be lysed.

3. Bacterial cellulose membranes can be prepared by the method of the present invention, and the bacteria that produce these bacterial cellulose membranes can achieve self-clearance through lysis without adding additional reagents. Therefore, the obtained bacterial cellulose powder is cleaner and pollution-free. , no residues of organic matter, etc., and is expected to achieve more uses.

4. The present invention solves the problems of self-clearance and release of produced intracellular substances by constructing a cleavage system in the host bacteria, with controllable conditions and high accuracy. It achieves more precise regulation of the cleavage protein by adjusting the RBS site.

Description of the drawings

Figure 1 is a blue light-controlled schematic diagram of the release of intracellular substances in Acetobacter xylinum and the detailed gene circuit in the engineered bacterium: Under dark conditions, the promoter pDawn is not turned on, and the engineered bacterium grows normally and generates BC membranes. When blue light is used, the pDawn promoter turns on the high expression of cleavage protein X174E. When its concentration reaches a certain threshold, Acetobacter xylin lyses and releases intracellular substances produced by Acetobacter xylinum.

Figure 2 shows the results of screening in Escherichia coli, in which: 1, 2, 3, and 4 respectively represent four different single clone spots on the LB agar plate. Only single clone point No. 4 can respond to blue light lysis and grow normally in the dark (its corresponding RBS is named RBS4).

Figure 3 shows the experimental results of blue light control of Acetobacter xylinum: the engineered bacteria can grow normally under light-proof conditions, but are completely lysed under light conditions.

Detailed ways

In order to make the above objects, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described in detail below, but this should not be understood as limiting the implementable scope of the present invention.

Example 1 Construction of Acetobacter xylinum capable of blue light controlled lysis

As illustrated in Figure 1, Acetobacter xylinum ATCC58532 was selected as the host bacterium to express the lytic protein through a plasmid. The vector plasmid selected pSEVA331. The blue light promoter pDawn (its sequence is shown in SEQ ID NO. 1) was used in the plasmid to control the phage φ174. Expression of cleavage protein E (X174E, sequence shown in SEQ ID NO. 2) resulted in pDawn-X174E-pSEVA331 plasmid. Finally, under dark conditions, the pDawn promoter in the engineered bacteria was not turned on, and the engineered bacteria grew normally and could produce cellulose membranes; however, when blue light (470nm) was used to illuminate the bacteria, pDawn in the engineered bacteria turned on the high expression of cleavage protein X174E, and the engineered bacteria Cleavage dies and releases intracellular substances.

However, under dark conditions, the promoter pDawn inevitably has leaky expression, so that the cleavage protein X174E is also produced at a low level. If the leaky expression of X174E protein is too high, the engineered bacteria will not be able to grow normally in the dark, resulting in the inability to obtain engineered bacteria containing the plasmid, or the engineered bacteria can grow normally in the dark, but cannot respond to blue light lysis. Therefore, the background expression level of X174E protein is the key to controllable lysis of engineered bacteria.

As mentioned earlier, in order to ensure that the engineered bacteria can grow and produce membranes normally under dark conditions, the concentration of the leaky expressed cleavage protein X174E must be lower than the threshold concentration required for lysis, and its concentration after the pDawn promoter is turned on The lysis threshold is reached. In order to obtain suitable engineering bacteria for the expression of X174E protein, the inventors adjusted the expression level of X174E protein by adjusting the sequence of the ribosome binding site before cleavage of the protein. At the same time, in order to improve the screening efficiency, preliminary screening was first conducted in E. coli, which has relatively mature molecular biology operations, and then the plasmids obtained from the preliminary screening were electroporated into Acetobacter xylinum for further screening. Specifically, random primer mutation method was first used to screen batches of E. coli TOP10.

The random primer method was used to construct the pDawn-RBSNNN-X174E-pSEVA331 series of plasmids: all fragments and vector connections in the experiment were obtained by Gibson assembly, and the primers designed by the random primer method were obtained by Shanghai Sangon Bioengineering Co., Ltd. synthesis. The pDawn-RBSNNN-X174E-pSEVA331 series of plasmid mixtures obtained by ligation using random primers were transferred into E. coli TOP10 supercompetent cells through chemical transformation, and finally plated in LB agar plates containing resistance and cultured overnight.

Randomly select a single colony spot from the agar plate that was cultured overnight and dissolve it in 20 μL of sterile water, and pipet 5 μL to spot two corresponding agar plates. After drying at room temperature, one of the plates was protected from light (wrapped in tin foil), and the other plate was placed under blue light (50 μW/cm ² ) and incubated for 16-24 hours.

The experimental results are shown in Figure 2. The experimental results show that different plasmids show different results. Screen for E. coli that can grow normally under light-proof conditions and can be completely lysed under blue light conditions.

Then, the appropriate plasmid screened from E. coli was subjected to second-generation sequencing and the RBS sequence was confirmed. The RBS sequence obtained by screening was shown in SEQ ID NO. 3 and was named RBS4. The appropriate plasmid selected, namely pDawn-RBS4-X174E-pSEVA331, was transformed into Acetobacter xylinum ATCC58532 by electroporation transformation (3KV, 2ms), spread on a resistant HS agar plate, and cultured for 4 days to finally obtain the project. strain pDawn-RBS4-X174E-pSEVA331-ATCC58532.

The experimental results are shown in Figure 3. Further experimental results show that pDawn-RBS4-X174E-pSEVA331-ATCC58532 can achieve normal growth under light-proof conditions and is completely lysed under blue light conditions.

In all experiments, LB broth was used as the growth medium for E. coli and Hestrin-Schramm (HS) medium was used for Acetobacter xylinum ATCC58532. If antibiotics need to be added, the concentrations of chloramphenicol (chl) used are 37 μg/mL and 148 μg/mL for Escherichia coli and Acetobacter xylinum respectively. In all experiments, unless otherwise stated, the culture temperatures of Escherichia coli and Acetobacter xylinum were 37°C and 30°C, respectively.

Components used and their corresponding sequences

pDawn SEQ ID NO.1

tcaatcgttgagcatgccggcgcgcatcgcgaggcgaaccagctccgaaaggctccgttggcctgcatcttggtcatgacgttggcccgatacacctcgatggtgcgcgggctgatgtcgtactcgcgggcgatcagcttgttggaaaggccggcgatcagcccttccatgacctggcgctcc ctggggctcaacgaggcgacgcgggcggcgatatcctgcgcgacggcctcgctcttggcggccggctcggcctggcggatcgccgattcgatcatggcggtgaggcggtcgtcctcgaaaggcttttccagaaagtccaccgcccctaacttcatcgcctcgaccgcgagcggcacgtcgccgtgaccgg tcatgatgaggatcggaaaggggctttgctgcgccttcatccgcttcaacagctcgatgccgtcaaggcccggcatgcgcacgtcggagacgacgcagccgaaggagagacccggcagggcgtcgagaaaggcttgcgcgtcgtcaaacagcgtgacgccgaagccggcagaatccagcaggaaattcag cgaatcccgcatcgccgcgtcgtcgtcgatgacgtagatatgtcccttggtcgtcatgctagacctcctatcatctcgtcggctgccgggagggtgaagcggaaggtcgccccgcccgatgcgttgctctcggcccacatgcgcccgccgtgagcttcgatgatcgagcggctgatggacagt cccacgcccatgccggtgtccttggtggtgaagaaagtctgaaacaggttcggaatgacgtcgtcctggaaaccgctgccggtgtcggacacttcgacctcgatcatgtcgtcggcggcgggggtgttggtgacgacgagctcgcgtcgctgcgactgagccatcgcttccagcgcg ttgcggaacaggttgaccaggacctgctggatctgcacccggtcggcgagaacgagatcggcgcccggatcgagactgaagcggagctgcacgttctgctcgcgcgcgccggcaagcccgagcgcgccggcctcctcgatcagcttggagagactctcgacccgcttctccgattcgccgcgggcaacga agtcgcgcaggcgccggatgatctggccggcgcgcagcgcctgctcggcggcgcggtccagggcgctttcgaccttcggtgtgtgttcggatcactgctgccggcaagcagccgccgcgagcccttcatgtagttgctgatcgccgccagcggctggttgagctcgtgcgcgagcgcggac gccatttcgcccatggcgctcagcctggagacgtggacgagctcggattgcagttcctggaggcgcgcctgggtctgctggtgctcggtgatatcattctgaataccgacaaaatacgttttatcctctatttccattggatcaatatttaattcattccagaacatcgttccgtcttttttgtagttttggatctgaacgg tgaccggttctttattttgtaaaagcggttctgatgttgtccacttctgcaggatctctgtgtgtttcccctgtaagaagcgacagttctttcctaaaatttcctcggtctcgtagccggtcatttgaacaaagccttgatttacgtagacaataggattatcttcaagtgcgggatctgtaattaccacacccgactc gcacgtgatcaagtgcttttttgatgacttccagctgtcctggtatcccaaatgattgaaaactagccacattcaccaccctcaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcaccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtag taggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtga tgccggccacgatgcgtccggcgtagaggatcgagatctacgcccgtgatcctgatcaccggctatccggacgaaaacatctcgacccgggccgccgaggccggcgtaaaagacgtggttttgaagccgcttctcgacgaaaacctgctcaagcgtatccgccgcgccatccaggaccggcctcgggcatgacct acggggttctacgtaaggcaccccccttaagatatcgctcgaaattttcgaacctcccgataccgcgtaccaatgcgtcatcacaacggagtctagaaaagaggagaaatactagatgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggc ttatcccaggaatctgtcgcagacaagatggggatggggcagtcaggcgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaagttagcgttgaagaatttagcccttcaatcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtcacttagaagtgag tatgagtaccctgtttttctcatgttcaggcagggatgttctcacctgagcttagaacctttacccaaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattctgcattctggcttgaggttgaaggtaattccatgaccgcaccaacaggctccaagccgagctttcctgacggaatgttaattctcg ttgaccctgagcaggctgttgagccaggtgatttctgcatagccagacttgggggtgatgagtttaccttcaagaaactgatcagggatagcggtcaggtgttttacaaccactaaacccacagtacccaatgatcccatgcaatgagagttgttccgttgtggggaaagttatcgctagtcagtggcctgaagagacgt ttggcgctgcaaacgacgaaaactacgctttagtagcttaataacgctgatagtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctg cgtttatatactagagtaacaccgtgcgtgttgactattttacctctggcggtgataatggttgc

X174E SEQ ID NO.2

atggtacgctggactttgtggggataccctcgctttcctgctcctgttgagtttattgctgccgtcattgcttattatgttcatcccgtcaacattcaaacggcctgtctcatcatggaaggcgctgaatttacggaaaacattattaatggcgtcgagcgtccggttaaagccgctgaattgttcgcgtttacct tgcgtgtacgcgcaggaaacactgacgttcttactgacgcagaagaaaacgtgcgtcaaaaattacgtgcggaaggagtga

RBS4 SEQ ID NO.3

tactagtgaacgatggcaaatactag.

SEQUENCE LISTING

<110> Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences

<120> A preparation method and method for controlling bacterial lysis and release of intracellular substances by physical stimulation to produce bacterial cellulose

application

<130> CP122010191C, PCT2201007

<160> 3

<170>PatentIn version 3.3

<210> 1

<211> 3369

<212> DNA

<213> Artificial sequence

<400> 1

tcaatcgttg agcatgccgg cgcgcatcgc gaggcgaacc agctccgaaa ggctgttggc 60

ctgcatcttg gtcatgacgt tggcccgata cacctcgatg gtgcgcgggc tgatgtcgta 120

ctcgcgggcg atcagcttgt tggaaaggcc ggcgatcagc ccttccatga cctggcgctc 180

cctggggctc aacgaggcga cgcgggcggc gatatcctgc gcgacggcct cgctcttggc 240

ggccggctcg gcctggcgga tcgccgattc gatcatggcg gtgaggcggt cgtcctcgaa 300

aggcttttcc agaaagtcca ccgcccctaa cttcatcgcc tcgaccgcga gcggcacgtc 360

gccgtgaccg gtcatgatga ggatcggaaa ggggctttgc tgcgccttca tccgcttcaa 420

cagctcgatg ccgtcaaggc ccggcatgcg cacgtcggag acgacgcagc cgaaggagag 480

acccggcagg gcgtcgagaa aggcttgcgc gtcgtcaaac agcgtgacgc cgaagccggc 540

agaatccagc aggaaattca gcgaatcccg catcgccgcg tcgtcgtcga tgacgtagat 600

atgtcccttg gtcgtcatgc tagacctcct atcatctcgt cggctgccgg gagggtgaag 660

cggaaggtcg ccccgcccga tgcgttgctc tcggcccaca tgcgcccgcc gtgagcttcg 720

atgatcgagc ggctgatgga cagtcccacg cccatgccgg tgtccttggt ggtgaagaaa 780

gtctgaaaca ggttcggaat gacgtcgtcc tggaaaccgc tgccggtgtc ggacacttcg 840

acctcgatca tgtcgtcggc ggcgggggtg ttggtgacga cgagctcgcg tcgctgcgac 900

tgagccatcg cttccagcgc gttgcggaac aggttgacca ggacctgctg gatctgcacc 960

cggtcggcga gaacgagatc ggcgccccgga tcgagactga agcggagctg cacgttctgc 1020

tcgcgcgcgc cggcaagccc gagcgcgccg gcctcctcga tcagcttgga gagactctcg 1080

acccgcttct ccgattcgcc gcgggcaacg aagtcgcgca ggcgccggat gatctggccg 1140

gcgcgcagcg cctgctcggc ggcgcggtcc agggcgcttt cgaccttcgg tgtgttcgga 1200

tcactgctgc cggcaagcag ccgccgcgag cccttcatgt agttgctgat cgccgccagc 1260

ggctggttga gctcgtgcgc gagcgcggac gccatttcgc ccatggcgct cagcctggag 1320

acgtggacga gctcggattg cagttcctgg aggcgcgcct gggtctgctg gtgctcggtg 1380

atatcattct gaataccgac aaaatacgtt ttatcctcta tttccattgg atcaatattt 1440

aattcattcc agaacatcgt tccgtctttt ttgtagtttt ggatctgaac ggtgaccggt 1500

tctttatttt gtaaagcggt tctgatgttg tccacttctg caggatctgt gtgtttcccc 1560

tgtaagaagc gacagttctt tcctaaaatt tcctcggtct cgtagccggt catttgaaca 1620

aagccttgat ttacgtagac aataggatta tcttcaagtg cgggatctgt aattaccaca 1680

ccgactcgca cgtgatcaag tgcttttttg atgacttcca gctgtcctgg tatcccaaat 1740

gattgaaaac tagccacatt caccaccctc aattgactct cttccgggcg ctatcatgcc 1800

ataccgcgaa aggttttgca ccattcgatg gtgtccggga tctcgacgct ctcccttatg 1860

cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc 1920

aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac 1980

catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc 2040

ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac 2100

gatgcgtccg gcgtagagga tcgagatcta cgcccgtgat cctgatcacc ggctatccgg 2160

acgaaaacat ctcgacccgg gccgccgagg ccggcgtaaa agacgtggtt ttgaagccgc 2220

ttctcgacga aaacctgctc aagcgtatcc gccgcgccat ccaggacgg cctcgggcat 2280

gacctacggg gttctacgta aggcaccccc cttaagatat cgctcgaaat tttcgaacct 2340

cccgataccg cgtaccaatg cgtcatcaca acggagtcta gaaaagagga gaaatactag 2400

atgagcacaa aaaagaaacc attaacacaa gagcagcttg aggacgcacg tcgccttaaa 2460

gcaatttatg aaaaaaagaa aaatgaactt ggctttatccc aggaatctgt cgcagacaag 2520

atggggatgg ggcagtcagg cgttggtgct ttatttaatg gcatcaatgc attaaatgct 2580

tataacgccg cattgcttgc aaaaattctc aaagttagcg ttgaagaatt tagcccttca 2640

atcgccagag aaatctacga gatgtatgaa gcggttagta tgcagccgtc acttagaagt 2700

gagtatgagt accctgtttt ttctcatgtt caggcaggga tgttctcacc tgagcttaga 2760

acctttacca aaggtgatgc ggagagatgg gtaagcacaa ccaaaaaagc cagtgattct 2820

gcattctggc ttgaggttga aggtaattcc atgaccgcac caacaggctc caagccgagc 2880

tttcctgacg gaatgttaat tctcgttgac cctgagcagg ctgttgagcc aggtgatttc 2940

tgcatagcca gacttggggg tgatgagttt accttcaaga aactgatcag ggatagcggt 3000

caggtgtttt tacaaccact aaacccacag tacccaatga tcccatgcaa tgagagttgt 3060

tccgttgtgg ggaaagttat cgctagtcag tggcctgaag agacgtttgg cgctgcaaac 3120

gacgaaaact acgctttagt agcttaataa cgctgatagt gctagtgtag atcgctacta 3180

gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct 3240

gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt 3300

tctgcgttta tatactagag taacaccgtg cgtgttgact attttacctc tggcggtgat 3360

aatggttgc 3369

<210> 2

<211> 276

<212> DNA

<213> Artificial sequence

<400> 2

atggtacgct ggactttgtg ggataccctc gctttcctgc tcctgttgag tttattgctg 60

ccgtcattgc ttattatgtt catcccgtca acattcaaac ggcctgtctc atcatggaag 120

gcgctgaatt tacggaaaac attattaatg gcgtcgagcg tccggttaaa gccgctgaat 180

tgttcgcgtt taccttgcgt gtacgcgcag gaaacactga cgttcttact gacgcagaag 240

aaaacgtgcg tcaaaaatta cgtgcggaag gagtga 276

<210> 3

<211> 26

<212> DNA

<213> Artificial sequence

<400> 3

tactagtgaa cgatggcaaa tactag 26

Claims (10)

1. A plasmid for physically controlling bacterial lysis, characterized in that the plasmid has a physically activated promoter and a gene encoding a lytic protein amplified under the control of the physically activated promoter, the gene encoding the lytic protein further has a ribosome binding site upstream, and the Ribosome Binding Site (RBS) has a sequence as set forth in SEQ ID No.3, or a sequence having a mutation of 1,2,3 or 4 bases in SEQ ID No. 3.

2. The plasmid for physical control bacterial lysis according to claim 1, wherein the physically activated promoter is a promoter capable of initiating initiation due to changes in light, temperature, pressure, osmotic pressure;

preferably, the light is preferably blue light;

preferably, the physically activated promoter is selected from the blue light promoter pDawn, more preferably, the sequence of which is shown in SEQ ID No. 1.

3. The plasmid for physically controlling bacterial lysis according to claim 1, characterized in that the lytic protein is selected from the group consisting of proteins capable of causing bacterial lysis, preferably the lytic protein E, LKD phage lytic protein of phage phi 174, lambda phage lytic protein;

more preferably, the coding sequence of the cleavage protein E of the phage phi 174 is shown in SEQ ID NO. 2.

4. A bacterial cellulose membrane-producing engineering bacterium having the plasmid for physical control lysis according to any one of claims 1 to 3, which is capable of being activated by physical stimulation to lyse.

5. The bacterial cellulose membrane-producing engineering bacterium according to claim 4, wherein the host bacterium for producing the bacterial cellulose membrane-producing engineering bacterium is at least one selected from the group consisting of Acetobacter xylinum, acetobacter pastoris, acetobacter xylosojae, gluconobacter hankii, acetobacter aceti, aerobacter, rhizobium, achromobacter, agrobacterium, pseudomonas, alcaligenes, sarcina, and Acetobacter.

6. The bacterial cellulose membrane-producing engineering bacterium according to claim 4 or 5, wherein the genome or plasmid of the bacterial cellulose membrane-producing engineering bacterium expresses a gene encoding an active substance;

preferably, the active substance is selected from the group consisting of proteins, RNAs, polypeptides.

7. The method for constructing a bacterial cellulose membrane-producing engineering bacterium according to any one of claims 4 to 6, comprising:

s11) constructing a plasmid for physically controlling bacterial lysis, wherein the plasmid is provided with a physically activated promoter and a coding gene of a lysate amplified under the control of the physically activated promoter, the coding gene of the lysate is provided with a Ribosome Binding Site (RBS) at the upstream of the coding gene of the lysate, and the Ribosome Binding Site (RBS) has a sequence shown in SEQ ID NO. 3;

s12) transferring the plasmid for physical control bacterial lysis into wild bacteria capable of generating bacterial cellulose membrane to obtain engineering bacteria capable of generating bacterial cellulose membrane capable of controlling lysis under physical conditions.

8. Use of a plasmid for physical control lysis according to any of claims 1-3 for the preparation of an engineered bacterium capable of being lysed by physical stimulation.

9. A method of modulating bacterial lysis and release of bacterial intracellular material, the method comprising:

s01) constructing the engineering bacteria capable of generating bacterial cellulose membrane according to any one of claims 4-6;

s02) activating physical stimulus to crack the bacterial cellulose membrane-forming engineering bacteria and release intracellular substances.

10. A method of constructing an engineered bacterium capable of inducing lysis by physical stimulation, the method comprising the steps of:

s1) selecting a corresponding promoter according to the physical stimulus type, and selecting a lysate according to the bacterial type;

s2) constructing a mixed plasmid connection liquid by adopting a random primer method, wherein the mixed plasmid connection liquid comprises the promoter selected in the step S1), a series of different ribosome binding sites designed and obtained by the random primer method and the coding gene sequence of the cracking protein selected in the step S1);

s3) transferring the mixed plasmid connection liquid obtained in the step S2) into escherichia coli to obtain escherichia coli engineering bacteria to be screened;

s4) respectively culturing the engineering bacteria of the escherichia coli to be screened under the physical stimulation in the step S1) and under the condition of non-physical stimulation, and screening to obtain escherichia coli strains which can normally grow under the condition of non-physical stimulation and are completely lysed under the condition of physical stimulation;

s5) extracting corresponding recombinant plasmids from the escherichia coli engineering bacteria obtained by screening in the step S4) and sequencing to obtain corresponding ribosome binding site sequences, and respectively introducing the recombinant plasmids into the wild bacteria in the step S1) to obtain engineering bacteria capable of being induced to crack by physical stimulation;

preferably, the bacteria are cellulose membrane-forming bacteria that can produce a foreign protein or target component;

preferably, the physical stimulus is a change in light, temperature, pressure, osmotic pressure.