WO2023010789A1 - Grna for targeted reduction of drug-resistant gene blatem and drug-resistant plasmid thereof, transferable knockout vector, and use thereof - Google Patents
Grna for targeted reduction of drug-resistant gene blatem and drug-resistant plasmid thereof, transferable knockout vector, and use thereof Download PDFInfo
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- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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Definitions
- the invention relates to the field of biotechnology, in particular to a gRNA targeting a bla TEM drug-resistant gene and a drug-resistant plasmid thereof, a horizontally transferable gene knockout vector and its application in reducing the bla TEM drug-resistant plasmid.
- Antibiotic resistance is often determined by drug resistance genes, which can be located on chromosomes and plasmids, and spread through gene vertical transfer and horizontal transfer. At present, drug resistance genes have been regarded as a new type of environmental pollutants.
- the drug resistance gene bla TEM is ubiquitous in the environment, and the broad-spectrum ⁇ -lactamase encoded by it can hydrolyze ampicillin and other ⁇ -lactam antibiotics, endowing bacteria with antibiotic resistance.
- the drug resistance gene bla TEM has been widely detected on multi-drug resistance plasmids, and these multi-drug resistance plasmids can often spread among bacteria of different species through conjugative transfer.
- pathogenic bacteria acquire these multidrug-resistant plasmids, it is difficult to be killed by corresponding antibiotics, which poses potential health risks to humans and animals.
- the existing antimicrobial resistance gene reduction technologies often change the bacterial community structure or directly kill the bacteria, for example, by changing the environmental conditions to change the bacterial community structure, thereby affecting the spread of multi-drug resistant plasmids, or killing bacteria through disinfection, etc.
- the method directly inactivates the bacteria, making it difficult for the donor bacteria to spread the multi-drug resistance plasmid to other bacteria.
- these methods did not essentially destroy the multidrug-resistant plasmids. Under suitable conditions, these multidrug-resistant plasmids carrying the drug-resistant gene bla TEM can still enter other bacteria through transformation and other methods, and continue to spread the drug-resistant genes.
- the reduction method using the CRISPR/Cas system to directly target knockout of multidrug-resistant plasmids can fundamentally overcome this problem.
- the use of horizontal transfer of plasmids to deliver knockout vectors can make the knockout process more sustainable and help to better control the migration and spread of multidrug-resistant plasmids.
- the present invention provides a gRNA for targeting and reducing the bla TEM drug-resistant gene and its drug-resistant plasmid, a transferable knockout vector and its application, specifically including targeting the drug-resistant gene bla TEM gRNA, horizontally transferable knockout vector, vector construction method and its application in the reduction of multidrug-resistant plasmids carrying bla TEM , this technology can be used to control the migration and spread of drug-resistant genes in the environment and other fields.
- the realization process of the present invention is as follows:
- the present invention provides targeted knockout of the drug resistance gene bla TEM and the gRNA sequence carrying the bla TEM multi-drug resistance plasmid, the sequence is as follows:
- gRNA TEM -1 5'-ATCGAACTGGATCTCAACAG-3'
- gRNA TEM -2 5'-ACAATTAATAGACTGGATGG-3'
- the present invention constructs a horizontally transferable gene knockout vector pCas9-Mob, which contains the knockout protein gene Cas9, gRNA expression sequence, horizontal transfer site gene, and the vector map is shown in Figure 1 .
- the present invention provides targeted knockout drug-resistant gene bla TEM and transferable knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 carrying bla TEM multidrug-resistant plasmids, the above
- the carrier maps are shown in Figure 2 and Figure 3 respectively.
- the above-mentioned knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 are characterized in that they can use specific Escherichia coli as a donor, and use the conjugation between bacteria to undergo horizontal transfer, and can also transform Enter the recipient bacteria, and target to knock out the corresponding site of bla TEM .
- the present invention provides a method for constructing the above-mentioned pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors, comprising the following steps:
- gRNA TEM -1 forward primer 5'-AAACATCGAACTGGATCTCAACAGG-3'
- gRNA TEM -1 reverse primer 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
- gRNA TEM -2 forward primer 5'-AAACACAATTAATAGACTGGATGGG-3'
- gRNA TEM -2 reverse primer 5'-AAAACCATCCAGTCTATTAATTGT-3'
- the construction method of the above-mentioned pCas9-Mob plasmid comprises the following steps:
- the transfer site of the plasmid was amplified by high-fidelity PCR technology, and OriT was obtained after gel purification.
- the above-mentioned pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors are transformed into competent cells of specific strains to obtain engineering strains carrying the above-mentioned knockout vectors, which are large intestine bacilli.
- the application of the above-mentioned pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 in reducing the drug-resistant gene bla TEM and the drug-resistant plasmid carrying bla TEM also falls within the protection scope of the present invention.
- microorganisms containing the above-mentioned knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 in the reduction of the drug-resistant gene bla TEM and the drug-resistant plasmid carrying bla TEM also belongs to the protection of the present invention scope.
- the present invention provides a gRNA targeting the drug-resistant gene bla TEM and carrying a bla TEM multidrug-resistant plasmid, which can guide the Cas9 protein to knock out the corresponding site.
- the transferable knockout vectors of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention can not only enter the recipient bacteria in the form of transformation, but also use specific Escherichia coli as a donor , using the conjugation between bacteria to produce horizontal transfer, can realize the knockout of the drug-resistant gene bla TEM and the multi-drug-resistant plasmid carrying bla TEM in a diversified way, and provide the drug-resistant gene bla TEM and multi-drug-resistant plasmid in the field of the environment
- the cuts are provided for technical reference.
- the present invention also provides methods for constructing pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors, which provide an effective reference and basis for knockout and reduction studies of other drug-resistant genes.
- gRNA TEM -1, gRNA TEM -2 and their knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention can effectively reduce drug resistance gene bla TEM and carry Multidrug resistance plasmid for bla TEM .
- it is easier to obtain the knockout vector targeting the antibiotic resistance gene bla TEM by using the preparation methods of the knockout vector pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention, and it is also useful for other environmental resistance.
- the research on the knockout and reduction of medicinal genes provides an effective method and basis.
- Figure 1 is the vector map of the knockout vector pCas9-Mob.
- Fig. 2 is a vector map of the knockout vector pCas9-Mob-gRNA TEM -1 involved in the present invention.
- Fig. 3 is a vector map of the knockout vector pCas9-Mob-gRNA TEM -2 involved in the present invention.
- Fig. 4 is the electropherogram of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors
- A pCas9-Mob-gRNA TEM -1 plasmid vector
- B pCas9-Mob-gRNA TEM -2 plasmid vector
- C linearized pCas9-Mob vector.
- Fig. 5 is the electrophoresis diagram of bla TEM gene cutting experiment in vitro
- A bla TEM gene
- B sgRNA TEM -1 cuts bla TEM gene
- C sgRNA TEM -2 cuts bla TEM gene
- cut fragments are in the dotted line box, and some original fragments may remain due to incomplete cutting due to too many initial fragments and short cutting time.
- Fig. 6 is a fluorescence characterization diagram of zygote colonies under a fluorescence microscope.
- A NK5449/bla TEM -mcherry colony
- B is the zygotic colony of the control plasmid pCas9-Mob
- C is the zygotic colony of pCas9-Mob-gRNA TEM -1
- D is the conjugation of pCas9-Mob-gRNA TEM -2 daughter colonies
- Figure 7 is a PCR gel electrophoresis picture of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 transformant colonies.
- the CaCl2 method was used to prepare competent cells, and the above-mentioned ligation product was transformed into a specific competent Escherichia coli, which integrated the relevant genes required for the horizontal transfer of the pCas9-Mob plasmid on the chromosome.
- the PCR reaction involved in Example 1 used Takara’s PrimeSTAR Max high-fidelity polymerase, and the reaction system was as follows: PrimeSTAR Max Premix 25ul, upstream and downstream primers 1ul each, template 1ul, ddH 2 O 22ul.
- the PCR reaction program was as follows: denaturation at 98°C for 10s, annealing at 55°C for 10s, extension temperature at 72°C, speed at 10s/kb, and cycle number at 30.
- the seamless cloning reaction system used in Example 1 is as follows: NovoRec plus recombinase 1ul, 5 ⁇ buffer 4ul, long fragment 0.03pmol, short fragment 0.05pmol, add the reaction system to 20ul with ddH 2 O, and react at 50°C 25 minutes.
- Example 2 Construction of a transferable knockout vector targeting the drug-resistant gene bla TEM and carrying a bla TEM multi-drug-resistant plasmid
- gRNA TEM -1 5'-ATCGAACTGGATCTCAACAG-3'
- gRNA TEM -2 5'-ACAATTAATAGACTGGATGG-3'
- gRNA TEM -1 forward primer 5'-AAACATCGAACTGGATCTCAACAGG-3'
- gRNA TEM -1 reverse primer 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
- gRNA TEM -2 forward primer 5'-AAACACAATTAATAGACTGGATGGG-3'
- gRNA TEM -2 reverse primer 5'-AAAACCATCCAGTCTATTAATTGT-3'
- connection system Ligate the digested pCas9-Mob vector and gRNA TEM -T1 and gRNA TEM -T2 respectively with T4 ligase to obtain the knockout vector pCas9 targeting the drug-resistant gene bla TEM and carrying the bla TEM multidrug-resistant plasmid -Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2.
- the connection system is as follows:
- knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 were verified by agarose electrophoresis.
- the above-mentioned knockout vectors were sequenced, and the sequences were verified to be correct, indicating that the vectors were constructed successfully.
- Example 4 Using fluorescent signals to verify the knockout effect of transferable knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2
- the plasmid bla TEM -mcherry carrying the bla TEM gene and the red fluorescent protein gene was transformed into Escherichia coli NK5449, the chromosome of which encodes Rif and Nal resistance, and the positive clone (NK5449/bla TEM -mcherry) was selected.
- Escherichia coli carrying the control vector pCas9-Mob, the knockout vector pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2, and the recipient bacteria carrying the bla TEM -mcherry plasmid were streaked and activated, respectively.
- step (3) Separately centrifuge the bacterial solution after the expanded culture in step (2) and discard the supernatant, add an appropriate amount of PBS buffer to the shaking tube to resuspend the bacterial solution that sinks to the bottom and centrifuge again, discard the supernatant again , and repeated 3 times. Finally, the OD600 value of the bacterial solution was adjusted to 0.7 ( ⁇ 0.05) with PBS buffer.
- the multi-drug resistance plasmid RP4 carrying bla TEM was transformed into Escherichia coli NK5449, the chromosome of which encodes Rif and Nal resistance, the positive clone (NK5449/RP4) was selected, and competent cells were prepared after LB amplification.
- gRNA TEM -1, gRNA TEM -2 and their knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention can effectively reduce drug resistance gene bla TEM and carry Multidrug resistance plasmid for bla TEM .
- knockout vector targeting antibiotic resistance gene bla TEM it is easier to obtain the knockout vector targeting antibiotic resistance gene bla TEM by using the preparation methods of the knockout vector pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention, and it is also for other drug resistance Gene knockout and reduction research provides an effective method and foundation, and this technology is helpful to control the migration and spread of drug resistance genes in the environment and other fields.
Abstract
A gRNA targeting a blaTEM drug-resistant gene and a drug-resistant plasmid carrying the blaTEM, a horizontally transferred gene knockout vector, and the use thereof in the reduction of the blaTEM drug-resistant plasmid. The provided gRNATEM-1, gRNATEM-2 and the knockout vectors pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2 can effectively reduce the blaTEM drug-resistant gene and the drug-resistant plasmid carrying the blaTEM. Moreover, by means of the provided method for preparing the knockout vectors pCas9-Mob-gRNATEM-1 and pCas9-Mob-gRNATEM-2, the knockout vectors targeting the blaTEM drug-resistant gene can be easily obtained. The method also provides an effective method and basis for the knockout and reduction research of other drug-resistant genes.
Description
本发明涉及生物技术领域，具体涉及靶向bla
TEM耐药基因及其耐药质粒的gRNA、一种可水平转移的基因敲除载体及其在bla
TEM耐药质粒削减方面的应用。
The invention relates to the field of biotechnology, in particular to a gRNA targeting a bla TEM drug-resistant gene and a drug-resistant plasmid thereof, a horizontally transferable gene knockout vector and its application in reducing the bla TEM drug-resistant plasmid.
近年来，随着抗生素的大量使用，越来越多的抗生素及其代谢产物被排放入污水处理系统甚至自然环境中，使得这些环境中的微生物所面临的选择性压力也日益增加。这些残留的抗生素会使得细菌不断适应并进化，进而获得抗生素耐药性。抗生素耐药性往往由耐药基因决定，这些耐药基因可位于染色体及质粒上，并通过基因垂直转移和水平转移等方式进行传播。目前，耐药基因已被视为一种新型的环境污染物。耐药基因bla
TEM在环境中普遍存在，它编码的广谱β-内酰胺酶可以使氨苄西林等β-内酰胺类抗生素水解，赋予细菌抗生素耐药性。目前，耐药基因bla
TEM在多重耐药质粒上被广泛检出，这些多重耐药质粒往往可以通过接合转移在不同种属的细菌间传播。当致病菌获得这些多重耐药质粒后，就难以被相应抗生素杀灭，进而对人类和动物产生潜在的健康风险。
In recent years, with the extensive use of antibiotics, more and more antibiotics and their metabolites have been discharged into the sewage treatment system and even the natural environment, making the microorganisms in these environments face increasing selective pressure. These residual antibiotics allow bacteria to continuously adapt and evolve to acquire antibiotic resistance. Antibiotic resistance is often determined by drug resistance genes, which can be located on chromosomes and plasmids, and spread through gene vertical transfer and horizontal transfer. At present, drug resistance genes have been regarded as a new type of environmental pollutants. The drug resistance gene bla TEM is ubiquitous in the environment, and the broad-spectrum β-lactamase encoded by it can hydrolyze ampicillin and other β-lactam antibiotics, endowing bacteria with antibiotic resistance. At present, the drug resistance gene bla TEM has been widely detected on multi-drug resistance plasmids, and these multi-drug resistance plasmids can often spread among bacteria of different species through conjugative transfer. When pathogenic bacteria acquire these multidrug-resistant plasmids, it is difficult to be killed by corresponding antibiotics, which poses potential health risks to humans and animals.
目前，现有的抗生素耐药基因的削减技术往往通过改变细菌群落结构或者直接杀灭细菌，例如，通过改变环境条件使得细菌群落结构改变，从而影响多重耐药质粒的传播，或者通过消毒等杀菌手段，将细菌直接灭活，使得供体细菌难以将多重耐药质粒传播到其他细菌。然而，这些方式并未从本质上破坏多重耐药质粒，在合适的条件下，这些携带耐药基因bla
TEM的多重耐药质粒仍可以通过转化等方式进入其他细菌，继续耐药基因的传播。因此，采用CRISPR/Cas系统直接靶向敲除多重耐药质粒的削减方法可以从根本上克服这一难题。同时，利用质粒的水平转移投递敲除载体可以使敲除过程更加可持续，有利于更好的控制多重耐药质粒的迁移传播。
At present, the existing antimicrobial resistance gene reduction technologies often change the bacterial community structure or directly kill the bacteria, for example, by changing the environmental conditions to change the bacterial community structure, thereby affecting the spread of multi-drug resistant plasmids, or killing bacteria through disinfection, etc. The method directly inactivates the bacteria, making it difficult for the donor bacteria to spread the multi-drug resistance plasmid to other bacteria. However, these methods did not essentially destroy the multidrug-resistant plasmids. Under suitable conditions, these multidrug-resistant plasmids carrying the drug-resistant gene bla TEM can still enter other bacteria through transformation and other methods, and continue to spread the drug-resistant genes. Therefore, the reduction method using the CRISPR/Cas system to directly target knockout of multidrug-resistant plasmids can fundamentally overcome this problem. At the same time, the use of horizontal transfer of plasmids to deliver knockout vectors can make the knockout process more sustainable and help to better control the migration and spread of multidrug-resistant plasmids.
发明内容Contents of the invention
为了解决现有技术存在的问题，本发明提供了靶向削减bla
TEM耐药基因及其耐药质粒的gRNA、一种可转移型敲除载体及其应用，具体包括靶向耐药基因bla
TEM的gRNA，可水平转移的敲除载体、载体构建方法及其在携带bla
TEM多重耐药质粒削减方面的应用，该技术可用于环境等领域耐药基因迁移传播的控制。
In order to solve the problems existing in the prior art, the present invention provides a gRNA for targeting and reducing the bla TEM drug-resistant gene and its drug-resistant plasmid, a transferable knockout vector and its application, specifically including targeting the drug-resistant gene bla TEM gRNA, horizontally transferable knockout vector, vector construction method and its application in the reduction of multidrug-resistant plasmids carrying bla TEM , this technology can be used to control the migration and spread of drug-resistant genes in the environment and other fields.
本发明的实现过程如下：The realization process of the present invention is as follows:
第一方面，本发明提供靶向敲除耐药基因bla
TEM及携带bla
TEM多重耐药质粒的gRNA序列，该序列如下所示：
In the first aspect, the present invention provides targeted knockout of the drug resistance gene bla TEM and the gRNA sequence carrying the bla TEM multi-drug resistance plasmid, the sequence is as follows:
gRNA
TEM-1：5’-ATCGAACTGGATCTCAACAG-3’
gRNA TEM -1: 5'-ATCGAACTGGATCTCAACAG-3'
gRNA
TEM-2：5’-ACAATTAATAGACTGGATGG-3’
gRNA TEM -2: 5'-ACAATTAATAGACTGGATGG-3'
第二方面，本发明构建了一种可水平转移的基因敲除载体pCas9-Mob，所述载体包含敲除蛋白基因Cas9，gRNA表达序列，水平转移位点基因，载体图谱如附图1所示。In the second aspect, the present invention constructs a horizontally transferable gene knockout vector pCas9-Mob, which contains the knockout protein gene Cas9, gRNA expression sequence, horizontal transfer site gene, and the vector map is shown in Figure 1 .
第三方面，本发明提供靶向敲除耐药基因bla
TEM及携带bla
TEM多重耐药质粒的可转移型敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2，上述载体图谱分别如附图2和附图3所示。
In a third aspect, the present invention provides targeted knockout drug-resistant gene bla TEM and transferable knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 carrying bla TEM multidrug-resistant plasmids, the above The carrier maps are shown in Figure 2 and Figure 3 respectively.
上述敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2特征在于，既能以特定大肠杆菌为供体，利用细菌间的接合作用发生水平转移，也可以通过转化的方式进入受体菌，靶向敲除bla
TEM相应位点。
The above-mentioned knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 are characterized in that they can use specific Escherichia coli as a donor, and use the conjugation between bacteria to undergo horizontal transfer, and can also transform Enter the recipient bacteria, and target to knock out the corresponding site of bla TEM .
第四方面，本发明提供上述pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2敲除载体的构建方法，包括如下步骤：
In a fourth aspect, the present invention provides a method for constructing the above-mentioned pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors, comprising the following steps:
(1)分别设计gRNA
TEM-1和gRNA
TEM-2的退火引物，序列如下：
(1) Design the annealing primers of gRNA TEM -1 and gRNA TEM -2 respectively, the sequences are as follows:
gRNA
TEM-1正向引物：5’-AAACATCGAACTGGATCTCAACAGG-3’
gRNA TEM -1 forward primer: 5'-AAACATCGAACTGGATCTCAACAGG-3'
gRNA
TEM-1反向引物：5’-AAAACCTGTTGAGATCCAGTTCGAT-3’
gRNA TEM -1 reverse primer: 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
gRNA
TEM-2正向引物：5’-AAACACAATTAATAGACTGGATGGG-3’
gRNA TEM -2 forward primer: 5'-AAACACAATTAATAGACTGGATGGG-3'
gRNA
TEM-2反向引物：5’-AAAACCCATCCAGTCTATTAATTGT-3’
gRNA TEM -2 reverse primer: 5'-AAAACCATCCAGTCTATTAATTGT-3'
(2)将上述正向引物和反向引物磷酸化并分别退火，纯化回收后得到靶向耐药基因bla
TEM及携带bla
TEM多重耐药质粒的gRNA双链，退火体系如下：
(2) Phosphorylate the above forward primer and reverse primer and anneal respectively. After purification and recovery, the gRNA duplex targeting the drug-resistant gene bla TEM and carrying the bla TEM multi-drug resistant plasmid is obtained. The annealing system is as follows:
在50ul的反应体系中，加入5ul T4 PNK缓冲液，5ul 10mM的ATP，1ul T4 PNK酶，100uM的正向与反向引物各1ul，最后用灭菌水补齐至50ul。37℃反应30min后，将温度上调至95℃，持续10min，再以1℃/min的速度缓慢降至室温。In the 50ul reaction system, add 5ul T4 PNK buffer, 5ul 10mM ATP, 1ul T4 PNK enzyme, 1ul each of 100uM forward and reverse primers, and finally make up to 50ul with sterilized water. After reacting at 37°C for 30 minutes, the temperature was raised to 95°C for 10 minutes, and then slowly lowered to room temperature at a rate of 1°C/min.
(3)用限制性核酸内酶切将上述pCas9-Mob载体线性化。(3) The above-mentioned pCas9-Mob vector was linearized by restriction endonuclease digestion.
(4)将酶切后的线性化pCas9-Mob载体和gRNA
TEM-T1、gRNA
TEM-T2退火双链分别用T4连接酶连接，得到靶向耐药基因bla
TEM及携带bla
TEM多重耐药质粒的可转移型敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2。连接体系如下：
(4) Ligate the digested linearized pCas9-Mob vector and the annealed double strands of gRNA TEM -T1 and gRNA TEM -T2 with T4 ligase to obtain the targeted drug-resistant gene bla TEM and the multidrug-resistant plasmid carrying bla TEM The transferable knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2. The connection system is as follows:
线性化pCas9-Mob片段0.05pmol，gRNA
TEM-T1或gRNA
TEM-T2退火双链0.15pmol，T4DNA连接酶1ul，连接缓冲液10ul，用无菌水补至20ul。反应条件为25℃孵育30min，反应完成后置于冰上。最后将反应液转化至感受态细胞中进行筛选并扩增。
Linearized pCas9-Mob fragment 0.05pmol, gRNA TEM-T1 or gRNA TEM-T2 annealed double strand 0.15pmol, T4 DNA ligase 1ul, ligation buffer 10ul, filled to 20ul with sterile water. The reaction condition was to incubate at 25°C for 30 min, and place on ice after the reaction was completed. Finally, the reaction solution was transformed into competent cells for screening and amplification.
第五方面，上述pCas9-Mob质粒的构建方法包括如下步骤：In the fifth aspect, the construction method of the above-mentioned pCas9-Mob plasmid comprises the following steps:
(1)以pCas9质粒为模板，利用高保真PCR技术扩增复制子OriV、筛选标记基因Chl以及敲除蛋白基因Cas9序列，凝胶纯化后获得OriV-Chl-Cas9。(1) Using the pCas9 plasmid as a template, high-fidelity PCR technology was used to amplify the replicon OriV, the screening marker gene Chl and the knockout protein gene Cas9 sequence, and obtain OriV-Chl-Cas9 after gel purification.
(2)以携带转移位点的质粒为模板，利用高保真PCR技术扩增该质粒的转移位点，凝胶纯化后获得OriT。(2) Using the plasmid carrying the transfer site as a template, the transfer site of the plasmid was amplified by high-fidelity PCR technology, and OriT was obtained after gel purification.
(3)利用无缝克隆技术连接上述纯化后的OriV-Chl-Cas9和OriT片段。(3) Utilize the seamless cloning technique to connect the above-mentioned purified OriV-Chl-Cas9 and OriT fragments.
(4)将上述连接产物转化入特定大肠杆菌，该大肠杆菌染色体上整合了pCas9-Mob质粒水平转移所需要的相关基因。(4) The above ligation product is transformed into specific Escherichia coli, and the relevant genes required for the horizontal transfer of the pCas9-Mob plasmid are integrated on the chromosome of the Escherichia coli.
(5)利用筛选标记Chl筛选出阳性克隆，将细菌扩大培养后提取质粒，该质粒即为敲除载体pCas9-Mob。(5) Use the screening marker Chl to screen out positive clones, expand the bacteria and extract the plasmid, which is the knockout vector pCas9-Mob.
第六方面，将上述pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2敲除载体转化入特定菌株的感受态细胞中，获得携带上述敲除载体的工程菌株，该菌株为大肠杆菌。
In the sixth aspect, the above-mentioned pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors are transformed into competent cells of specific strains to obtain engineering strains carrying the above-mentioned knockout vectors, which are large intestine bacilli.
第七方面，上述pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2在耐药基因bla
TEM及携带bla
TEM 耐药质粒削减方面的应用也属于本发明的保护范围。
In the seventh aspect, the application of the above-mentioned pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 in reducing the drug-resistant gene bla TEM and the drug-resistant plasmid carrying bla TEM also falls within the protection scope of the present invention.
第八方面，含有上述敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2的微生物在耐药基因bla
TEM及携带bla
TEM耐药质粒削减方面应用也属于本发明的保护范围。
In the eighth aspect, the use of microorganisms containing the above-mentioned knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 in the reduction of the drug-resistant gene bla TEM and the drug-resistant plasmid carrying bla TEM also belongs to the protection of the present invention scope.
本发明的有益效果至少包括：The beneficial effects of the present invention at least include:
(1)本发明通过序列分析及实验验证，提供了靶向耐药基因bla
TEM及携带bla
TEM多重耐药质粒的gRNA，该gRNA能引导Cas9蛋白敲除相应位点。
(1) Through sequence analysis and experimental verification, the present invention provides a gRNA targeting the drug-resistant gene bla TEM and carrying a bla TEM multidrug-resistant plasmid, which can guide the Cas9 protein to knock out the corresponding site.
(2)本发明提供的pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2可转移型敲除载体既能以转化的方式进入受体菌，也能以特定大肠杆菌为供体，利用细菌间的接合作用发生水平转移，能以多元化的途径实现耐药基因bla
TEM及携带bla
TEM多重耐药质粒的敲除，为环境等领域中耐药基因bla
TEM及多重耐药质粒的削减提供技术参考。
(2) The transferable knockout vectors of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention can not only enter the recipient bacteria in the form of transformation, but also use specific Escherichia coli as a donor , using the conjugation between bacteria to produce horizontal transfer, can realize the knockout of the drug-resistant gene bla TEM and the multi-drug-resistant plasmid carrying bla TEM in a diversified way, and provide the drug-resistant gene bla TEM and multi-drug-resistant plasmid in the field of the environment The cuts are provided for technical reference.
(3)本发明还提供了pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2敲除载体的构建方法，为其它耐药基因的敲除及削减研究提供了有效参考和基础。
(3) The present invention also provides methods for constructing pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors, which provide an effective reference and basis for knockout and reduction studies of other drug-resistant genes.
综上所述，本发明提供的gRNA
TEM-1、gRNA
TEM-2及其敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2可以有效削减耐药基因bla
TEM及携带bla
TEM的多重耐药质粒。同时，利用本发明提供的敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2制备方法较易得到靶向抗生素耐药基因bla
TEM的敲除载体，也为其他环境耐药基因的敲除及削减研究提供了有效方法和基础。
In summary, gRNA TEM -1, gRNA TEM -2 and their knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention can effectively reduce drug resistance gene bla TEM and carry Multidrug resistance plasmid for bla TEM . At the same time, it is easier to obtain the knockout vector targeting the antibiotic resistance gene bla TEM by using the preparation methods of the knockout vector pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention, and it is also useful for other environmental resistance. The research on the knockout and reduction of medicinal genes provides an effective method and basis.
图1为敲除载体pCas9-Mob的载体图谱。Figure 1 is the vector map of the knockout vector pCas9-Mob.
图2为本发明所涉及的敲除载体pCas9-Mob-gRNA
TEM-1的载体图谱。
Fig. 2 is a vector map of the knockout vector pCas9-Mob-gRNA TEM -1 involved in the present invention.
图3为本发明所涉及的敲除载体pCas9-Mob-gRNA
TEM-2的载体图谱。
Fig. 3 is a vector map of the knockout vector pCas9-Mob-gRNA TEM -2 involved in the present invention.
图4为pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2敲除载体的电泳图，
Fig. 4 is the electropherogram of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors,
其中，A：pCas9-Mob-gRNA
TEM-1质粒载体；B：pCas9-Mob-gRNA
TEM-2质粒载体；C：线性化pCas9-Mob载体。
Among them, A: pCas9-Mob-gRNA TEM -1 plasmid vector; B: pCas9-Mob-gRNA TEM -2 plasmid vector; C: linearized pCas9-Mob vector.
图5为bla
TEM基因体外切割实验电泳图，
Fig. 5 is the electrophoresis diagram of bla TEM gene cutting experiment in vitro,
其中，A：bla
TEM基因；B：sgRNA
TEM-1切割bla
TEM基因；C：sgRNA
TEM-2切割bla
TEM基因
Among them, A: bla TEM gene; B: sgRNA TEM -1 cuts bla TEM gene; C: sgRNA TEM -2 cuts bla TEM gene
注：虚线框内为切割后片段，部分原片段残留可能是由于初始片段多，切割时间短等导致未完全切割。Note: The cut fragments are in the dotted line box, and some original fragments may remain due to incomplete cutting due to too many initial fragments and short cutting time.
图6为荧光显微镜下接合子菌落荧光表征图。Fig. 6 is a fluorescence characterization diagram of zygote colonies under a fluorescence microscope.
其中A：NK5449/bla
TEM-mcherry菌落；B为对照质粒pCas9-Mob的接合子菌落；C为pCas9-Mob-gRNA
TEM-1的接合子菌落；D为pCas9-Mob-gRNA
TEM-2的接合子菌落；
Among them, A: NK5449/bla TEM -mcherry colony; B is the zygotic colony of the control plasmid pCas9-Mob; C is the zygotic colony of pCas9-Mob-gRNA TEM -1; D is the conjugation of pCas9-Mob-gRNA TEM -2 daughter colonies;
图7为pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2转化子菌落PCR凝胶电泳图。
Figure 7 is a PCR gel electrophoresis picture of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 transformant colonies.
实施例1可水平转移的基因敲除载体pCas9-Mob的构建及验证Example 1 Construction and verification of horizontally transferable gene knockout vector pCas9-Mob
(1)以pCas9质粒为模板，设计特异性引物，利用高保真PCR技术扩增复制子、筛选标记基因以 及敲除蛋白基因Cas9序列，凝胶纯化后获得OriV-Chl-Cas9。(1) Using the pCas9 plasmid as a template, design specific primers, use high-fidelity PCR technology to amplify the replicon, screening marker gene and knockout protein gene Cas9 sequence, and obtain OriV-Chl-Cas9 after gel purification.
(2)以携带转移位点的质粒为模板，设计特异性引物，利用高保真PCR技术扩增该质粒的转移位点，凝胶纯化后获得OriT。(2) Using the plasmid carrying the transfer site as a template, design specific primers, use high-fidelity PCR technology to amplify the transfer site of the plasmid, and obtain OriT after gel purification.
(3)采用Novoprotein无缝克隆试剂，依照protocol连接上述纯化后OriV-Chl-Cas9和OriT片段。(3) Using the Novoprotein seamless cloning reagent, connect the above-mentioned purified OriV-Chl-Cas9 and OriT fragments according to the protocol.
(4)利用CaCl
2法制备感受态，将上述连接产物转化入特定大肠杆菌感受态，该大肠杆菌染色体上整合了pCas9-Mob质粒水平转移所需要的相关基因。
(4) The CaCl2 method was used to prepare competent cells, and the above-mentioned ligation product was transformed into a specific competent Escherichia coli, which integrated the relevant genes required for the horizontal transfer of the pCas9-Mob plasmid on the chromosome.
(5)利用氯霉素筛选标记筛选出阳性克隆，细菌扩大培养后提取质粒，并保存菌株。(5) Use the chloramphenicol selection marker to screen out positive clones, extract the plasmid after the bacteria are expanded and cultured, and store the strain.
(6)对pCas9-Mob进行测序，经验证后序列无误，表明该载体构建成功。(6) The pCas9-Mob was sequenced, and the sequence was verified to be correct, indicating that the vector was constructed successfully.
实施例1中涉及的PCR反应采用Takara公司的PrimeSTAR Max高保真聚合酶，反应体系如下：PrimeSTAR Max Premix 25ul，上下游引物各1ul，模板1ul，ddH
2O 22ul。PCR反应程序如下：98℃变性10s，55℃退火10s，延伸温度为72℃，速度为10s/kb，循环数为30。
The PCR reaction involved in Example 1 used Takara’s PrimeSTAR Max high-fidelity polymerase, and the reaction system was as follows: PrimeSTAR Max Premix 25ul, upstream and downstream primers 1ul each, template 1ul, ddH 2 O 22ul. The PCR reaction program was as follows: denaturation at 98°C for 10s, annealing at 55°C for 10s, extension temperature at 72°C, speed at 10s/kb, and cycle number at 30.
实施例1中采用的无缝克隆反应体系如下：NovoRec plus重组酶1ul，5×缓冲液4ul，长片段0.03pmol，短片段为0.05pmol，用ddH
2O将反应体系补充至20ul，50℃反应25分钟。
The seamless cloning reaction system used in Example 1 is as follows: NovoRec plus recombinase 1ul, 5× buffer 4ul, long fragment 0.03pmol, short fragment 0.05pmol, add the reaction system to 20ul with ddH 2 O, and react at 50°C 25 minutes.
实施例2构建靶向耐药基因bla
TEM及携带bla
TEM多重耐药质粒的可转移型敲除载体
Example 2 Construction of a transferable knockout vector targeting the drug-resistant gene bla TEM and carrying a bla TEM multi-drug-resistant plasmid
(1)从环境来源的耐药质粒上获得耐药基因bla
TEM的序列，检索PAM位点，从紧邻PAM位点的gRNA中筛选出GC比，特异性等指标符合要求的gRNA，一般以20-25bp为宜。本案例优选的gRNA为：
(1) Obtain the sequence of the drug-resistant gene bla TEM from the drug-resistant plasmid derived from the environment, search for the PAM site, and screen out the gRNA that meets the requirements for GC ratio and specificity from the gRNA adjacent to the PAM site, generally at 20 -25bp is suitable. The preferred gRNA in this case is:
gRNA
TEM-1：5’-ATCGAACTGGATCTCAACAG-3’
gRNA TEM -1: 5'-ATCGAACTGGATCTCAACAG-3'
gRNA
TEM-2：5’-ACAATTAATAGACTGGATGG-3’
gRNA TEM -2: 5'-ACAATTAATAGACTGGATGG-3'
(2)分别设计gRNA
TEM-1和gRNA
TEM-2所涉及的退火引物，序列如下：
(2) Design the annealing primers involved in gRNA TEM -1 and gRNA TEM -2 respectively, the sequences are as follows:
gRNA
TEM-1正向引物：5’-AAACATCGAACTGGATCTCAACAGG-3’
gRNA TEM -1 forward primer: 5'-AAACATCGAACTGGATCTCAACAGG-3'
gRNA
TEM-1反向引物：5’-AAAACCTGTTGAGATCCAGTTCGAT-3’
gRNA TEM -1 reverse primer: 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'
gRNA
TEM-2正向引物：5’-AAACACAATTAATAGACTGGATGGG-3’
gRNA TEM -2 forward primer: 5'-AAACACAATTAATAGACTGGATGGG-3'
gRNA
TEM-2反向引物：5’-AAAACCCATCCAGTCTATTAATTGT-3’
gRNA TEM -2 reverse primer: 5'-AAAACCATCCAGTCTATTAATTGT-3'
(3)将上述正向引物和反向引物磷酸化并分别退火，纯化回收后得到靶向耐药基因bla
TEM及其耐药质粒的gRNA双链，分别为gRNA
TEM-T1和gRNA
TEM-T2，退火体系如下：
(3) Phosphorylate and anneal the above forward primer and reverse primer respectively, and obtain gRNA double strands targeting the drug-resistant gene bla TEM and its drug-resistant plasmid after purification and recovery, respectively gRNA TEM -T1 and gRNA TEM -T2 , the annealing system is as follows:
在50ul的反应体系中，加入5ul T4 PNK缓冲液，5ul 10mM的ATP，1ul T4 PNK酶，100uM的正向与反向引物各1ul，最后用灭菌水补齐至50ul。37℃反应30min后，将温度上调至95℃，持续10min，再以1℃/min的速度缓慢降至室温。In the 50ul reaction system, add 5ul T4 PNK buffer, 5ul 10mM ATP, 1ul T4 PNK enzyme, 1ul each of 100uM forward and reverse primers, and finally make up to 50ul with sterilized water. After reacting at 37°C for 30 minutes, the temperature was raised to 95°C for 10 minutes, and then slowly lowered to room temperature at a rate of 1°C/min.
(4)凝胶回收实例1中涉及的经限制性内酶酶切后的pCas9-Mob载体。(4) Gel recovery of the pCas9-Mob vector involved in Example 1 after digestion with restriction enzymes.
(5)将酶切后的pCas9-Mob载体和gRNA
TEM-T1、gRNA
TEM-T2分别用T4连接酶连接，得到靶向耐药基因bla
TEM及携带bla
TEM多重耐药质粒的敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2。连接体系如下：
(5) Ligate the digested pCas9-Mob vector and gRNA TEM -T1 and gRNA TEM -T2 respectively with T4 ligase to obtain the knockout vector pCas9 targeting the drug-resistant gene bla TEM and carrying the bla TEM multidrug-resistant plasmid -Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2. The connection system is as follows:
线性化pCas9-Mob片段0.05pmol，gRNA
TEM-T1或gRNA
TEM-T2退火双链0.15pmol，T4DNA连接酶1ul，连接缓冲液10ul，用无菌水补至20ul。反应条件为25℃孵育30min，反应完成后置于冰上。最后将 反应液转化至感受态细胞中。
Linearized pCas9-Mob fragment 0.05pmol, gRNA TEM- T1 or gRNA TEM -T2 annealed double strand 0.15pmol, T4 DNA ligase 1ul, ligation buffer 10ul, filled to 20ul with sterile water. The reaction condition was to incubate at 25°C for 30 min, and place on ice after the reaction was completed. Finally, the reaction solution was transformed into competent cells.
(6)采用琼脂糖电泳对敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2的大小进行验证，胶图如附图4所示，结果表明大小符合预期。分别对上述敲除载体进行测序，经验证后序列无误，表明该载体构建成功。
(6) The sizes of the knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 were verified by agarose electrophoresis. The above-mentioned knockout vectors were sequenced, and the sequences were verified to be correct, indicating that the vectors were constructed successfully.
实施例3利用体外靶向切割实验验证gRNA
TEM-1和gRNA
TEM-2对bla
TEM的有效性
Example 3 Validation of the effectiveness of gRNA TEM -1 and gRNA TEM -2 on bla TEM using in vitro targeted cleavage experiments
(1)设计并合成正向引物gRNA
TEM-1-F和gRNA
TEM-2-F，序列如下：
(1) Design and synthesize forward primers gRNA TEM -1-F and gRNA TEM -2-F, the sequences are as follows:
gRNA
TEM-1-F：
gRNA TEM -1-F:
5’-TTAATACGACTCACTATAGGATCGAACTGGATCTCAACAGGTTTTAGAGCTAGAAATAG-3’5'-TTAATACGACTCACTATAGGATCGAACTGGATCTCAACAGGTTTTTAGAGCTAGAAATAG-3'
gRNA
TEM-2-F：
gRNA TEM -2-F:
5’-TTAATACGACTCACTATAGGACAATTAATAGACTGGATGGGTTTTAGAGCTAGAAATAG-3’5'-TTAATACGACTCACTATAGGACAATTAATAGACTGGATGGGTTTTTAGAGCTAGAAATAG-3'
(2)在20ul体系中加入10ul 2×sgRNA反应缓冲液，2ul上述正向引物，2ul酶混合液，6ul无菌水，体外转录1h，得到sgRNA
TEM-1和sgRNA
TEM-2。
(2) Add 10ul of 2×sgRNA reaction buffer, 2ul of the above forward primer, 2ul of enzyme mixture, 6ul of sterile water into the 20ul system, transcribe in vitro for 1h, and obtain sgRNA TEM -1 and sgRNA TEM -2.
(3)取适量纯化后的bla
TEM基因片段，加入2ul反应缓冲液，1ul Cas9酶混合液，1ul上述sgRNA
TEM，并用无菌水补至20ul，37℃反应1h，70℃反应10min。
(3) Take an appropriate amount of purified bla TEM gene fragments, add 2ul of reaction buffer, 1ul of Cas9 enzyme mixture, 1ul of the above sgRNA TEM , make up to 20ul with sterile water, react at 37°C for 1h, and react at 70°C for 10min.
(4)将上述bla
TEM基因片段、经sgRNA
TEM-1和sgRNA
TEM-2的切割后bla
TEM基因片段用1％的琼脂糖凝胶进行电泳实验，结果如附图5所示。结果表明，gRNA
TEM-1和gRNA
TEM-2对bla
TEM有靶向切割作用。
(4) The above bla TEM gene fragment and the bla TEM gene fragment cut by sgRNA TEM -1 and sgRNA TEM -2 were subjected to electrophoresis experiments with 1% agarose gel, and the results are shown in Fig. 5 . The results showed that gRNA TEM -1 and gRNA TEM -2 had targeted cleavage of bla TEM .
实施例4利用荧光信号验证可转移型敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2的敲除效果
Example 4 Using fluorescent signals to verify the knockout effect of transferable knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2
(1)将携带bla
TEM基因和红色荧光蛋白基因的质粒bla
TEM-mcherry转入大肠杆菌NK5449，该菌染色体编码Rif和Nal抗性，挑选阳性克隆(NK5449/bla
TEM-mcherry)。
(1) The plasmid bla TEM -mcherry carrying the bla TEM gene and the red fluorescent protein gene was transformed into Escherichia coli NK5449, the chromosome of which encodes Rif and Nal resistance, and the positive clone (NK5449/bla TEM -mcherry) was selected.
(2)将携带对照载体pCas9-Mob、敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2的大肠杆菌、携带bla
TEM-mcherry质粒的受体菌划线活化后分别接种于相应抗性的LB液体培养基中，置于恒温震荡培养箱中37℃震荡培养16小时，速度为200rmp。
(2) Escherichia coli carrying the control vector pCas9-Mob, the knockout vector pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2, and the recipient bacteria carrying the bla TEM -mcherry plasmid were streaked and activated, respectively. Inoculated in the corresponding resistant LB liquid medium, placed in a constant temperature shaking incubator at 37°C for shaking culture for 16 hours at a speed of 200rmp.
(3)将步骤(2)扩大培养后的菌液分别离心后弃上清液，向摇菌管中加入适量PBS缓冲液使沉底的菌液重悬并再次离心，再次弃去上清液，共重复3次。最后，用PBS缓冲液将菌液OD600值均调节至0.7(±0.05)。(3) Separately centrifuge the bacterial solution after the expanded culture in step (2) and discard the supernatant, add an appropriate amount of PBS buffer to the shaking tube to resuspend the bacterial solution that sinks to the bottom and centrifuge again, discard the supernatant again , and repeated 3 times. Finally, the OD600 value of the bacterial solution was adjusted to 0.7 (±0.05) with PBS buffer.
(4)实验组：在10ml离心管中加入2.5ml PBS重悬的携带bla
TEM-mcherry质粒的受体菌与2.5ml PBS重悬的含有pCas9-Mob-gRNA
TEM-1敲除载体的大肠杆菌或含有pCas9-Mob-gRNA
TEM-2敲除载体的大肠杆菌。对照组：在10ml离心管中加入2.5ml PBS重悬的携带bla
TEM-mcherry质粒的受体菌与2.5ml PBS重悬的含有pCas9-Mob载体的大肠杆菌。
(4) Experimental group: add 2.5ml PBS resuspended recipient bacteria carrying blaTEM -mcherry plasmid and 2.5ml PBS resuspended Escherichia coli containing pCas9-Mob-gRNA TEM -1 knockout vector in a 10ml centrifuge tube Or E. coli containing the pCas9-Mob-gRNA TEM -2 knockout vector. Control group: add 2.5ml PBS resuspended recipient bacteria carrying blaTEM -mcherry plasmid and 2.5ml PBS resuspended Escherichia coli containing pCas9-Mob vector in a 10ml centrifuge tube.
(5)37℃培养16h后，取步骤(4)中实验组和对照组的菌液各100ul涂布于Nal、Rif、Chl三种抗性的LB固体培养基上，筛选接合子。(5) After culturing at 37° C. for 16 hours, take 100 ul of the bacterial solutions from the experimental group and the control group in step (4) and spread them on the LB solid medium with three resistances of Nal, Rif, and Chl to screen zygotes.
(6)采用荧光体式显微镜观察LB平板上的菌落，若bla
TEM基因被敲除，则同一质粒上的红色荧光蛋白基因也消失，菌落不发荧光；反之，则发荧光。
(6) Observe the colonies on the LB plate with a fluorescent microscope. If the bla TEM gene is knocked out, the red fluorescent protein gene on the same plasmid will also disappear, and the colonies will not fluoresce; otherwise, they will fluoresce.
(7)实验结果如附图6所示，pCas9-Mob的接合子菌落发红色荧光，pCas9-Mob-gRNA
TEM-1和 pCas9-Mob-gRNA
TEM-2的接合子菌落无明显荧光，表明可水平转移型载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2能高效敲除携带bla
TEM基因的耐药质粒。
(7) The experimental results are shown in Figure 6, the zygotic colonies of pCas9-Mob emit red fluorescence, and the zygotic colonies of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 have no obvious fluorescence, indicating that The horizontal transfer vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 can efficiently knock out the drug-resistant plasmid carrying the bla TEM gene.
实施例5利用菌落PCR验证pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2对多重耐药质粒的敲除效果
Example 5 Verification of the knockout effect of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 on multidrug-resistant plasmids by colony PCR
(1)将携带bla
TEM的多重耐药质粒RP4转入大肠杆菌NK5449，该菌染色体编码Rif和Nal抗性，挑选阳性克隆(NK5449/RP4)，LB扩增后制备感受态细胞。
(1) The multi-drug resistance plasmid RP4 carrying bla TEM was transformed into Escherichia coli NK5449, the chromosome of which encodes Rif and Nal resistance, the positive clone (NK5449/RP4) was selected, and competent cells were prepared after LB amplification.
(2)将等量pCas9-Mob、pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2载体分别转化入100ul上述感受态细胞，静置30min后，42℃水浴60秒，然后转移至冰上，冷却5分钟。向冷却后的感受态细胞中加入1ml LB液体培养基，混匀后置于37℃，200rpm摇床中复苏60分钟。吸取复苏后的感受态细胞100ul涂布于添加Nal、Rif、Chl三种抗性的LB培养基，筛选转化子，置于恒温培养箱中培养15小时。
(2) Transform equal amounts of pCas9-Mob, pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 vectors into 100ul of the above-mentioned competent cells respectively, after standing for 30min, put them in a 42°C water bath for 60 seconds, and then transfer Transfer to ice and chill for 5 minutes. Add 1ml of LB liquid medium to the cooled competent cells, mix well and place in a shaker at 37°C and 200rpm for 60 minutes to recover. 100ul of recovered competent cells were sucked and spread on LB medium supplemented with Nal, Rif, and Chl three kinds of resistances, and transformants were screened, and cultured in a constant temperature incubator for 15 hours.
(3)依据RP4质粒的序列设计鉴定引物，分别挑选pCas9-Mob、pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2的转化子各5株，将菌落稀释后进行PCR实验。若RP4质粒被敲除，则PCR结果不会出现明显亮条带，反之，则存在明显亮条带。
(3) Design and identify primers based on the sequence of the RP4 plasmid, select 5 transformants each of pCas9-Mob, pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2, and perform PCR experiments after dilution of the colonies. If the RP4 plasmid is knocked out, there will be no obvious bright bands in the PCR results, otherwise, there will be obvious bright bands.
(4)实验结果如附图7所示，pCas9-Mob转化子条带明显，pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2的转化子无明显亮条带。因此，pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2敲除载体可以有效削减携带bla
TEM的多重耐药质粒。
(4) The experimental results are shown in Figure 7, the pCas9-Mob transformants have obvious bands, but the transformants of pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 have no obvious bright bands. Therefore, pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 knockout vectors can effectively reduce the multi-drug resistance plasmid carrying bla TEM .
综上所述，本发明提供的gRNA
TEM-1、gRNA
TEM-2及其敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2可以有效削减耐药基因bla
TEM及携带bla
TEM的多重耐药质粒。同时，利用本发明提供的敲除载体pCas9-Mob-gRNA
TEM-1和pCas9-Mob-gRNA
TEM-2制备方法较易得到靶向抗生素耐药基因bla
TEM的敲除载体，也为其他耐药基因的敲除及削减研究提供了有效方法和基础，该技术有助于环境等领域耐药基因迁移传播的控制。
In summary, gRNA TEM -1, gRNA TEM -2 and their knockout vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention can effectively reduce drug resistance gene bla TEM and carry Multidrug resistance plasmid for bla TEM . At the same time, it is easier to obtain the knockout vector targeting antibiotic resistance gene bla TEM by using the preparation methods of the knockout vector pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 provided by the present invention, and it is also for other drug resistance Gene knockout and reduction research provides an effective method and foundation, and this technology is helpful to control the migration and spread of drug resistance genes in the environment and other fields.
Claims (10)
- 特异性靶向抗生素耐药基因bla TEM及携带bla TEM耐药质粒的gRNA，其特征在于，其编码的核苷酸序列为gRNA TEM-1：5’-ATCGAACTGGATCTCAACAG-3’，gRNA TEM-2：5’-ACAATTAATAGACTGGATGG-3’。 Specifically targeting antibiotic resistance gene bla TEM and gRNA carrying bla TEM drug resistance plasmid, characterized in that the nucleotide sequence encoded by it is gRNA TEM -1: 5'-ATCGAACTGGATCTCAACAG-3', gRNA TEM -2: 5'-ACAATTAATAGACTGGATGG-3'.
- 一种可水平转移的敲除载体pCas9-Mob，其特征在于，所述载体包含敲除蛋白基因Cas9，gRNA表达序列，水平转移位点OriT。A horizontally transferable knockout vector pCas9-Mob is characterized in that the vector comprises a knockout protein gene Cas9, a gRNA expression sequence, and a horizontal transfer site OriT.
- 含有权利要求1所述特异性靶向耐药基因bla TEM及携带bla TEM多重耐药质粒的gRNA TEM-1、gRNA TEM-2的可转移型基因敲除载体pCas9-Mob-gRNA TEM-1或pCas9-Mob-gRNA TEM-2。 The transferable gene knockout vector pCas9-Mob-gRNA TEM-1 containing the specific targeting drug resistance gene bla TEM described in claim 1 and the gRNA TEM -1 and gRNA TEM- 2 carrying the bla TEM multi-drug resistance plasmid or pCas9-Mob-gRNA TEM -2.
- 一种构建权利要求3所述的基因敲除载体pCas9-Mob-gRNA TEM-1或pCas9-Mob-gRNA TEM-2的方法，其特征在于，包括如下步骤： A method for constructing the gene knockout carrier pCas9-Mob-gRNA TEM -1 or pCas9-Mob-gRNA TEM -2 according to claim 3, comprising the steps of:(1)根据权利要求1所述的gRNA TEM-1和gRNA TEM-2序列设计退火引物，分别为： (1) according to gRNA TEM -1 and gRNA TEM -2 sequence design annealing primers according to claim 1, are respectively:gRNA TEM-1正向引物：5’-AAACATCGAACTGGATCTCAACAGG-3’ gRNA TEM -1 forward primer: 5'-AAACATCGAACTGGATCTCAACAGG-3'gRNA TEM-1反向引物：5’-AAAACCTGTTGAGATCCAGTTCGAT-3’ gRNA TEM -1 reverse primer: 5'-AAAACCTGTTGAGATCCAGTTCGAT-3'gRNA TEM-2正向引物：5’-AAACACAATTAATAGACTGGATGGG-3’ gRNA TEM -2 forward primer: 5'-AAACACAATTAATAGACTGGATGGG-3'gRNA TEM-2反向引物：5’-AAAACCCATCCAGTCTATTAATTGT-3’ gRNA TEM -2 reverse primer: 5'-AAAACCATCCAGTCTATTAATTGT-3'(2)将gRNA TEM-1和gRNA TEM-2的正向引物和反向引物分别退火，形成双链，并用T4 PNK酶磷酸化； (2) Anneal the forward and reverse primers of gRNA TEM -1 and gRNA TEM -2, respectively, to form double strands, and phosphorylate them with T4 PNK enzyme;(3)利用无缝克隆技术将质粒水平转移位点OriT插入pCas9质粒中，构建可转移的pCas9-Mob质粒，利用核酸内切酶将其线性化，并凝胶回收；(3) Insert the plasmid horizontal transfer site OriT into the pCas9 plasmid by using seamless cloning technology to construct a transferable pCas9-Mob plasmid, linearize it with endonuclease, and gel recover;(4)利用T4连接酶将磷酸化后的gRNA TEM-1和gRNA TEM-2退火双链分别与线性化pCas9-Mob载体连接，得到的载体即为权利要求3所述的可转移型敲除载体pCas9-Mob-gRNA TEM-1和pCas9-Mob-gRNA TEM-2。 (4) Ligate the phosphorylated gRNA TEM -1 and gRNA TEM -2 annealed double strands to the linearized pCas9-Mob carrier using T4 ligase, and the obtained carrier is the transferable knockout described in claim 3 Vectors pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2.
- 根据权利要求4所述的方法，其特征在于：所述步骤(3)pCas9-Mob质粒的构建方法包括如下步骤：method according to claim 4, is characterized in that: the construction method of described step (3) pCas9-Mob plasmid comprises the steps:(1)以pCas9质粒为模板，利用高保真PCR技术扩增复制子OriV、筛选标记基因Chl以及敲除蛋白基因Cas9序列，凝胶纯化后获得OriV-Chl-Cas9；(1) Using the pCas9 plasmid as a template, high-fidelity PCR technology was used to amplify the replicon OriV, the screening marker gene Chl and the knockout protein gene Cas9 sequence, and obtain OriV-Chl-Cas9 after gel purification;(2)以携带转移位点的质粒为模板，利用高保真PCR技术扩增该质粒的转移位点，凝胶纯化后获得OriT；(2) Using the plasmid carrying the transfer site as a template, use high-fidelity PCR technology to amplify the transfer site of the plasmid, and obtain OriT after gel purification;(3)利用无缝克隆技术连接上述纯化后的OriV-Chl-Cas9和OriT片段；(3) Utilize the seamless cloning technique to connect the above-mentioned purified OriV-Chl-Cas9 and OriT fragments;(4)将上述连接产物转化入特定大肠杆菌，该大肠杆菌染色体上整合了pCas9-Mob质粒水平转移所需要的相关基因；(4) transforming the above ligation product into specific Escherichia coli, which integrates the relevant genes required for the horizontal transfer of the pCas9-Mob plasmid on the chromosome of the Escherichia coli;(5)利用筛选标记Chl筛选出阳性克隆，将细菌扩大培养后提取质粒，该质粒即为转移型敲除载体pCas9-Mob。(5) Use the screening marker Chl to screen out positive clones, expand the bacteria and extract the plasmid, which is the transfer knockout vector pCas9-Mob.
- 权利要求1所述的gRNA TEM-1或gRNA TEM-2在抗生素耐药基因bla TEM及携带bla TEM的多重耐药质粒敲除及削减方面的应用。 The application of the gRNA TEM -1 or gRNA TEM -2 described in claim 1 in knocking out and reducing the antibiotic resistance gene bla TEM and the multi-drug resistance plasmid carrying bla TEM .
- 权利要求3或4所述的基因敲除载体pCas9-Mob-gRNA TEM-1或pCas9-Mob-gRNA TEM-2在抗生素耐药基因bla TEM及携带bla TEM的多重耐药质粒敲除及削减方面的应用。 The gene knockout vector pCas9-Mob-gRNA TEM -1 or pCas9-Mob-gRNA TEM -2 described in claim 3 or 4 is knocked out and reduced in terms of antibiotic resistance gene bla TEM and multi-drug resistance plasmid carrying bla TEM Applications.
- 权利要求3或4所述的敲除载体pCas9-Mob-gRNA TEM-1或pCas9-Mob-gRNA TEM-2，其特征在于，可以通过转化或水平转移两种方式进入受体微生物。 The knockout vector pCas9-Mob-gRNA TEM -1 or pCas9-Mob-gRNA TEM -2 according to claim 3 or 4, is characterized in that it can enter the recipient microorganism through transformation or horizontal transfer.
- 含有权利要求3或4所述的敲除载体pCas9-Mob-gRNA TEM-1和pCas9-Mob-gRNA TEM-2的微生物，该微生物为大肠杆菌。 The microorganism containing the knockout carrier pCas9-Mob-gRNA TEM -1 and pCas9-Mob-gRNA TEM -2 described in claim 3 or 4, the microorganism is Escherichia coli.
- 权利要求9所述微生物在耐药基因bla TEM及携带bla TEM的多重耐药质粒敲除及削减方面的应用。 The application of the microorganism described in claim 9 in knocking out and reducing the drug resistance gene bla TEM and the multi-drug resistance plasmid carrying bla TEM .
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| CN105463003A (en) * | 2015-12-11 | 2016-04-06 | 扬州大学 | Recombinant vector for eliminating activity of kanamycin drug resistance gene and building method of recombinant vector |
| CN107384926A (en) * | 2017-08-13 | 2017-11-24 | 中国人民解放军疾病预防控制所 | A kind of CRISPR Cas9 systems for targetting bacteria removal Drug Resistance Plasmidss and application |
| CN108135949A (en) * | 2015-08-14 | 2018-06-08 | 内梅西斯生物有限公司 | Delivery vehicle |
| CN111378660A (en) * | 2020-02-29 | 2020-07-07 | 浙江大学 | sgRNA targeting tetracycline resistance gene tetA, knockout vector thereof, vector construction method and application |
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| CN110423772B (en) * | 2019-07-17 | 2023-04-21 | 上海科技大学 | Cytosine base editing plasmid for Acinetobacter baumannii and application of cytosine base editing plasmid |
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| CN105463003A (en) * | 2015-12-11 | 2016-04-06 | 扬州大学 | Recombinant vector for eliminating activity of kanamycin drug resistance gene and building method of recombinant vector |
| CN107384926A (en) * | 2017-08-13 | 2017-11-24 | 中国人民解放军疾病预防控制所 | A kind of CRISPR Cas9 systems for targetting bacteria removal Drug Resistance Plasmidss and application |
| WO2021119594A1 (en) * | 2019-12-13 | 2021-06-17 | The Regents Of The University Of California | Reducing antibiotic resistance in bacteria using pro-active genetics |
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