WO2023197093A1 - 一种物理刺激控制可生成细菌纤维素细菌裂解和胞内物质释放的制备方法和应用 - Google Patents

一种物理刺激控制可生成细菌纤维素细菌裂解和胞内物质释放的制备方法和应用 Download PDF

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WO2023197093A1
WO2023197093A1 PCT/CN2022/086016 CN2022086016W WO2023197093A1 WO 2023197093 A1 WO2023197093 A1 WO 2023197093A1 CN 2022086016 W CN2022086016 W CN 2022086016W WO 2023197093 A1 WO2023197093 A1 WO 2023197093A1
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lysis
bacteria
plasmid
protein
bacterial cellulose
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高艳梅
金帆
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中国科学院深圳先进技术研究院
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  • 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.
  • 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.
  • BC bacterial cellulose
  • 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 also tried to use Acetobacter xylin to express some foreign proteins, but the problem of controlled release of intracellularly expressed proteins and other substances has not yet been solved [4] .
  • 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.
  • 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 the sequence described in SEQ ID NO.3.
  • the physically activated promoter is a promoter that can be initiated due to changes in light, temperature, pressure, and osmotic pressure.
  • the light is preferably blue light.
  • the physically activated promoter is selected from the blue light promoter pDawn, preferably, its sequence is shown in SEQ ID NO.1.
  • the lytic protein is selected from proteins that can cause bacterial lysis, preferably lytic protein E of phage ⁇ 174, LKD16 phage lytic protein, and lambda phage lytic protein.
  • the coding sequence of the lytic protein E of the phage ⁇ 174 is shown in SEQ ID NO. 2.
  • 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.
  • 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.
  • the genome or plasmid of the engineered bacteria that can produce bacterial cellulose membranes can express genes encoding exogenous active substances.
  • 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;
  • 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:
  • 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:
  • 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.
  • step S3 Transfer the mixed plasmid ligation solution obtained in step S2) into E. coli to obtain the E. coli engineering strain to be screened;
  • step 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.
  • step 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.
  • bacteria are bacteria that can produce cellulose membranes that can produce exogenous proteins or target components.
  • the physical stimulus is changes in light, temperature, pressure, and osmotic pressure.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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 ⁇ 174.
  • Expression of cleavage protein E (X174E, the sequence is shown in SEQ ID NO. 2), the pDawn-X174E-pSEVA331 plasmid was obtained.
  • 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.
  • 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.
  • the inventors adjusted the expression level of X174E protein by adjusting the sequence of the ribosome binding site before cleavage of the protein.
  • 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 all provided 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.
  • 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.
  • 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 is 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.
  • LB broth was used as the growth medium for E. coli and Hestrin-Schramm (HS) medium was used for Acetobacter xylinum ATCC58532.
  • HS Hestrin-Schramm
  • concentrations of chloramphenicol (chl) used are 37 ⁇ g/mL and 148 ⁇ g/mL for Escherichia coli and Acetobacter xylinum respectively.
  • the culture temperatures of Escherichia coli and Acetobacter xylinum were 37°C and 30°C, respectively.

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Abstract

提供了一种物理刺激控制可生成细菌纤维素工程菌裂解的制备方法和应用，一种物理控制细菌裂解用质粒，所述质粒中具有物理激活启动子以及受物理激活启动子调控扩增的裂解蛋白的编码基因，所述裂解蛋白的编码基因上游还具有核糖体结合位点，所述核糖体结合位点(RBS)具有如SEQ ID NO.3所述的序列，或者SEQ ID NO.3上具有1个、2个、3个或4个碱基突变的序列。通过在宿主菌内构建裂解系统实现了其自我清除和胞内物质释放问题，条件可控、准确度高，通过调节裂解蛋白的RBS位点实现对工程菌裂解的精准的调控。

Description

一种物理刺激控制可生成细菌纤维素细菌裂解和胞内物质释放的制备方法和应用 技术领域
本发明属于生物技术领域，具体涉及一种蓝光控制的可生成纤维素膜的木醋杆菌裂解死亡和胞内物质释放系统。
背景技术
细菌生长速度快，便于大规模培养，因此早就被研究者们用来生产各种药物。随着合成生物学地发展，利用工程化后的细菌作为药物输送系统具有传统给药方法无比比拟的优点 [1]。比如，细菌可以保护药物的活性，增加药物的半衰期；更重要的是，细菌可以将药物输送到注射或口服难以到达的身体部位。
相比其他细菌，木醋杆菌等微生物可以生产一种强壮和超纯的天然细菌纤维素(BC)。而BC具有良好的生物相容性、能够为细胞附着提供最佳的三维基质、对多种细胞无毒性、能够提供灵活性、高保水能力和气体交换 [2][3]。虽然BC膜已经被广泛的应用于食品，医疗等领域，但是对木醋杆菌菌株本身的改造却少之又少，使得其功能较为单一。近年来，也有研究者尝试利用木醋杆菌来表达一些外源蛋白，但是胞内表达的蛋白等物质的可控释放问题并仍未解决 [4]。
[1]Kang M,Choe D,Kim K,et al.Synthetic Biology Approaches in The Development of Engineered Therapeutic Microbes[J].Int J Mol Sci,2020,21(22):
[2]Picheth GF,Pirich CL,Sierakowski MR,et al.Bacterial cellulose in biomedical 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 of bacterial 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.
发明内容
为解决上述问题，本发明构建了一种物理激活的裂解系统来控制可生成细菌纤维素膜的细菌自我清除以及胞内物质的释放。本发明通过合成生物学的方法，在可生成细菌纤维素的细菌内构建一套受物理刺激调控的裂解系统来远程控制可生成细菌纤维素的细菌裂解和胞内物质释放。工程化后的可生成细菌纤维素的细菌在黑暗条件下可以正常生长和生成BC膜， 但当使用物理刺激后可生成细菌纤维素的细菌发生裂解同时释放出胞内生成的物质。
本发明一个方面提供了一种物理控制细菌裂解用质粒，所述质粒中具有物理激活启动子以及受物理激活启动子调控扩增的裂解蛋白的编码基因，所述裂解蛋白的编码基因上游还具有核糖体结合位点，所述核糖体结合位点(ribosome binding site，RBS)具有如SEQ ID NO.3所述的序列。
进一步地，所述物理激活启动子为能够由于光、温度、压力、渗透压变化引发启动的启动子。
进一步地，所述光优选为蓝光。
进一步地，所述物理激活启动子选自蓝光启动子pDawn，优选地，其序列如SEQ ID NO.1所示。
进一步地，所述裂解蛋白选自能够导致细菌裂解的蛋白，优选为噬菌体φ174的裂解蛋白E、LKD16噬菌体裂解蛋白，λ噬菌体裂解蛋白。
进一步地，所述噬菌体φ174的裂解蛋白E的编码序列如SEQ ID NO.2所示。
进一步地，质粒选自在可生成细菌纤维素的细菌中可复制的任意质粒，例如以pSEVA331作为载体质粒。
本发明另一个方面提供了一种可生成细菌纤维素膜(BC)工程菌，所述可生成细菌纤维素膜工程菌具有本发明上述物理控制裂解用质粒，能够被物理刺激激活裂解。
进一步地，所述生产细菌纤维素膜工程菌的宿主菌选自木醋杆菌、巴氏醋杆菌、木葡糖酸醋杆菌、汉氏葡糖醋杆菌、醋化醋杆菌、产醋醋杆菌、气杆菌、根瘤杆菌、无色杆菌、土壤杆菌、假单胞杆菌、产碱杆菌、八叠球菌、动胶菌中的至少一种。
进一步地，所述可生成细菌纤维素膜的工程菌的基因组或质粒可表达外源活性物质的编码基因。
进一步地，所述活性物质选自蛋白质、RNA、多肽。
本发明又一个方面提供了可生成细菌纤维素膜工程菌的构建方法，所述构建方法包括：
S11)构建物理控制细菌裂解用质粒，所述质粒中具有物理激活启动子以及受物理激活启动子调控扩增的裂解蛋白的编码基因，所述裂解蛋白的编码基因上游还具有核糖体结合位点，所述核糖体结合位点具有如SEQ ID NO.3所述的序列；
S12)将所述物理控制细菌裂解用质粒转入可生成细菌纤维素膜的野生型细菌中，获得能够以物理条件控制裂解的生产细菌纤维素膜工程菌。
本发明再一个方面提供了本发明所述的物理控制裂解用质粒在制备能够受物理刺激裂解 的细菌中的用途。
本发明再一个方面提供了一种调控细菌裂解以及释放细菌所产胞内物质的方法，所述方法包括：
S01)构建上述可生成细菌纤维素膜的工程菌；
S02)启动物理刺激激活上述可生成细菌纤维素膜工程菌裂解。
本发明再一个方面提供了一种能够被物理刺激诱导裂解的工程菌的构建方法，所述方法包括以下步骤：
S1)根据物理刺激种类选择对应的启动子，并根据细菌种类选择裂解蛋白；
S2)采用随机引物法构建混合的质粒连接液，所述混合的质粒连接液中包含步骤S1)选择的启动子、由随机引物法设计获得的一系列不同的核糖体结合位点以及由步骤S1)选择的裂解蛋白的编码基因序列；
S3)将步骤S2)获得的混合的质粒连接液转入大肠杆菌中，获得待筛选大肠杆菌工程菌；
S4)将待筛选大肠杆菌工程菌分别在步骤S1)所述物理刺激下，以及非所述物理刺激条件下分别进行培养，筛选能够获得能够在非所述物理刺激条件下正常生长，而在所述物理刺激条件下全部裂解的大肠杆菌菌株；
S5)从步骤S4)筛选获得的大肠杆菌工程菌中提取对应的重组质粒并进行测序，获得其对应的核糖体结合位点序列，并将该重组质粒分别导入步骤S1)所述野生型细菌中，获得能够被物理刺激诱导裂解的工程菌。
进一步地，所述细菌为可以生成外源蛋白或目标成分的可生成纤维素膜的细菌
进一步地，所述物理刺激为光、温度、压力、渗透压的变化。
有益效果
1、本发明提供了一种使用非侵入型诱导方式来控制工程菌裂解的方式，避免了化学诱导剂的侵入以及在细菌纤维素膜中扩散问题，同时这种调控方式不受时间和空间限制。
2、本发明通过特殊方法解决了裂解蛋白漏表达，导致在未受到物理刺激时，工程菌不会因为裂解蛋白的漏表达而裂解，进而无法得到足够的纤维素膜或期望的蛋白，或者并未在希望裂解的时间实现裂解宿主菌的效果。同时本发明还解决了裂解蛋白在受到物理刺激启动后无法达到裂解最低阈值水平，而无法实现宿主菌的裂解。
3、通过本发明的方法可以制备得到细菌纤维素膜，且生产这些细菌纤维素膜的细菌无需添加额外的试剂就能通过裂解实现自我清除，因此，获得的细菌纤维素末更干净，无污染，无有机物等的残留，有望实现更多用途。
4、本发明通过在宿主菌内构建裂解系统实现了其自我清除和所生产胞内物质释放问题，条件可控、准确度高，通过调节RBS位点实现裂解蛋白更加精准的调控。
附图说明
图1为蓝光控制的木醋杆菌胞内物质释放和工程菌内详细的基因回路示意图：在黑暗条件启动子pDawn不开启，工程菌正常生长和生成BC膜。当使用蓝光光照后，pDawn启动子开启裂解蛋白X174E的高表达，当其浓度达到一定阈值后裂解木醋杆菌释放出木醋杆菌所生产的胞内物质。
图2为大肠杆菌内筛选结果图，其中：1，2，3，4分别代表LB琼脂板四个不同的单克隆点。只有4号单克隆点可以响应蓝光裂解同时避光条件下正常生长(其对应RBS命名为RBS4)。
图3为蓝光控制木醋杆菌实验结果图：工程菌在避光条件下可以正常生长，但是光照条件完全裂解。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1构建可蓝光控制裂解的木醋杆菌
结合图1说明，选用木醋杆菌ATCC58532菌为宿主菌，通过质粒表达裂解蛋白，载体质粒选择pSEVA331，质粒中使用蓝光启动子pDawn(其序列如SEQ ID NO.1所示)来控制噬菌体φ174的裂解蛋白E(X174E，序列如SEQ ID NO.2所示)的表达，得到pDawn-X174E-pSEVA331质粒。最终在黑暗条件下，工程菌内pDawn启动子不开启，工程菌正常生长并且可以生成纤维素膜；但是当使用蓝光(470nm)光照后，工程菌内pDawn开启裂解蛋白X174E的高表达，工程菌裂解死亡并且可释放出胞内物质。
但是，在黑暗条件下启动子pDawn不可避免地存在漏表达，使的裂解蛋白X174E也被低水平地生产。如果X174E蛋白漏表达过高的话，工程菌在黑暗条件下也无法正常生长进而导致无法得到含有质粒的工程菌，或者工程菌在黑暗情况下可以正常生长，但是也无法响应蓝光裂解。因此，X174E蛋白的背景表达量是工程菌裂解可控的关键。
如前面所述，为了保证工程菌可以在黑暗条件正常生长和产膜，那么其漏表达的裂解蛋白X174E的浓度就必须低于其裂解所需的阈值浓度，并且在pDawn启动子开启后其浓度达到 裂解阈值。为了得到X174E蛋白表达合适的工程菌，本发明人通过调整裂解蛋白前核糖体结合位点的序列来调整X174E蛋白的表达量。同时，为了提高筛选效率，先在分子生物学操作较为成熟的大肠杆菌中进行初步筛选，然后再将初步筛选得到的质粒电转入木醋杆菌中进一步筛选。具体地，先采用随机引物突变法先在大肠杆菌TOP10中批量筛选。
随机引物法构建pDawn-RBSNNN-X174E-pSEVA331系列质粒的方法为：实验中所有片段和载体连接均通过吉布森连接法(Gibson assembly)得到，随机引物法设计的引物均由上海生工生物工程有限公司合成。通过使用随机引物法连接得到pDawn-RBSNNN-X174E-pSEVA331系列质粒混合液通过化学转化法转入大肠杆菌TOP10超级感受态中，并最终涂板在含有抗性的LB琼脂板中过夜培养。
从过夜培养的琼脂板中随机挑选单克隆点溶于20μL无菌水中，吸5μL分别点两块对应的琼脂板。室温晾干后，其中一块板避光处理(用锡纸包裹)，另一块板置于蓝光(50μW/cm 2)下培养16-24小时。
实验结果见图2，实验结果显示，不同的质粒显示了不同的结果，筛选在避光条件下可以正常生长，且在蓝光条件下能够全部裂解的大肠杆菌。
然后将大肠杆菌中筛选出的合适的质粒经过二代测序，并确认RBS序列，筛选获得的RBS序列如SEQ ID NO.3所示，命名为RBS4。将筛选出的合适质粒，即pDawn-RBS4-X174E-pSEVA331通过电击转化(3KV，2ms)的方法转入木醋杆菌ATCC58532中，涂布于含有抗性的HS琼脂板中培养4天最终得到工程菌pDawn-RBS4-X174E-pSEVA331-ATCC58532。
实验结果见图3，进一步实验结果显示，pDawn-RBS4-X174E-pSEVA331-ATCC58532能够实现在避光条件下正常生长，而在蓝光条件下全部裂解。
在所有实验中，大肠杆菌均使用LB肉汤用作生长培养基，木醋杆菌ATCC58532均使用Hestrin-Schramm(HS)培养基用作生长培养基。如需要添加抗生素，对大肠杆菌和木醋杆菌，氯霉素(chl)使用浓度分别为37μg/mL和148μg/mL。所有的实验中，如非特别说明大肠杆菌和木醋杆菌培养温度分别为37℃和30℃。
所用元件及其对应序列
pDawn SEQ ID NO.1
Figure PCTCN2022086016-appb-000001
Figure PCTCN2022086016-appb-000002
X174E SEQ ID NO.2
Figure PCTCN2022086016-appb-000003
RBS4 SEQ ID NO.3
Figure PCTCN2022086016-appb-000004

Claims (10)

  1. 一种物理控制细菌裂解用质粒，其特征在于，所述质粒中具有物理激活启动子以及受物理激活启动子调控扩增的裂解蛋白的编码基因，所述裂解蛋白的编码基因上游还具有核糖体结合位点，所述核糖体结合位点(RBS)具有如SEQ ID NO.3所述的序列，或者SEQ ID NO.3上具有1个、2个、3个或4个碱基突变的序列。
  2. 权利要求1所述的物理控制细菌裂解用质粒，其特征在于，所述物理激活启动子为能够由于光、温度、压力、渗透压变化引发启动的启动子；
    优选地，所述光优选为蓝光；
    优选地，所述物理激活启动子选自蓝光启动子pDawn，更优选地，其序列如SEQ ID NO.1所示。
  3. 权利要求1所述的物理控制细菌裂解用质粒，其特征在于，所述裂解蛋白选自能够导致细菌裂解的蛋白，优选为噬菌体φ174的裂解蛋白E、LKD16噬菌体裂解蛋白，λ噬菌体裂解蛋白；
    更优选地，所述噬菌体φ174的裂解蛋白E的编码序列如SEQ ID NO.2所示。
  4. 一种可生成细菌纤维素膜工程菌，其特征在于，所述可生成细菌纤维素膜工程菌具有权利要求1-3任一项所述的物理控制裂解用质粒，其能够被物理刺激激活裂解。
  5. 权利要求4所述的可生成细菌纤维素膜工程菌，其特征在于，所述生产细菌纤维素膜工程菌的宿主菌选自木醋杆菌、巴氏醋杆菌、木葡糖酸醋杆菌、汉氏葡糖醋杆菌、醋化醋杆菌、产醋醋杆菌、气杆菌、根瘤杆菌、无色杆菌、土壤杆菌、假单胞杆菌、产碱杆菌、八叠球菌、动胶菌中的至少一种。
  6. 权利要求4或5所述的可生成细菌纤维素膜工程菌，其特征在于，所述生产细菌纤维素膜的工程菌的基因组或者质粒表达了能够产生活性物质的编码基因；
    优选地，所述活性物质选自蛋白质、RNA、多肽。
  7. 权利要求4-6任一项所述可生成细菌纤维素膜工程菌的构建方法，所述构建方法包括：
    S11)构建物理控制细菌裂解用质粒，所述质粒中具有物理激活启动子以及受物理激活启动子调控扩增的裂解蛋白的编码基因，所述裂解蛋白的编码基因上游还具有核糖体结合位点，所述核糖体结合位点(RBS)具有如SEQ ID NO.3所述的序列；
    S12)将所述物理控制细菌裂解用质粒转入可生成细菌纤维素膜的野生型细菌中，获得能够以物理条件控制裂解的可生成细菌纤维素膜工程菌。
  8. 权利要求1-3任一项所述的物理控制裂解用质粒在制备能够受物理刺激裂解的工程细菌中的用途。
  9. 一种调控细菌裂解以及释放细菌胞内物质的方法，所述方法包括：
    S01)构建权利要求4-6任一项所述的可生成细菌纤维素膜的工程菌；
    S02)启动物理刺激激活上述可生成细菌纤维素膜工程菌裂解以及胞内物质释放。
  10. 一种能够被物理刺激诱导裂解的工程菌的构建方法，所述方法包括以下步骤：
    S1)根据物理刺激种类选择对应的启动子，并根据细菌种类选择裂解蛋白；
    S2)采用随机引物法构建混合的质粒连接液，所述混合的质粒连接液中包含步骤S1)选择的启动子、由随机引物法设计获得的一系列不同的核糖体结合位点以及由步骤S1)选择的裂解蛋白的编码基因序列；
    S3)将步骤S2)获得的混合的质粒连接液转入大肠杆菌中，获得待筛选大肠杆菌工程菌；
    S4)将待筛选大肠杆菌工程菌分别在步骤S1)所述物理刺激下，以及非所述物理刺激条件下分别进行培养，筛选能够获得能够在非所述物理刺激条件下正常生长，而在所述物理刺激条件下全部裂解的大肠杆菌菌株；
    S5)从步骤S4)筛选获得的大肠杆菌工程菌中提取对应的重组质粒并进行测序，获得其对应的核糖体结合位点序列，并将该重组质粒分别导入步骤S1)所述野生型细菌中，获得能够被物理刺激诱导裂解的工程菌；
    优选地，所述细菌为可以生成外源蛋白或目标成分的可生成纤维素膜的细菌；
    优选地，所述物理刺激为光、温度、压力、渗透压的变化。
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