CN116218751A - Method for knocking out staphylococcus aureus SdrC protein gene - Google Patents
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a method for knocking out a staphylococcus aureus SdrC protein gene, which comprises the steps of designing primers of upstream and downstream homologous arm sequences of the SdrC protein according to a genome sequence of the staphylococcus aureus, amplifying a fusion fragment of staphylococcus aureus RN4220 by overlapping PCR, constructing an sdrC recombinant suicide plasmid, transforming a vector, constructing a deletion mutant, verifying a final knocked-out strain by sequencing and the like. According to the method for knocking out the staphylococcus aureus SdrC protein gene, the SdrC recombinant suicide plasmid is constructed, the knocking-out vector is constructed, the RN4220 wild strain is electrically transformed from the vector, the deletion mutant strain is constructed, finally, the SdrC amino acid fragment with the same functional annotation is successfully knocked out, the formation of a biological film mediated by the SdrC protein is inhibited, and the infection of the staphylococcus aureus to a host is slowed down.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for knocking out a staphylococcus aureus SdrC protein gene.
Background
Staphylococcus aureus (Staphylococcus aureus), a gram-positive (g+) conditional pathogen widely present in the human population, can cause a series of diseases such as osteomyelitis. The staphylococcus aureus SdrC protein has the main function of promoting the formation of a staphylococcus aureus biological film through the dimerization of the protein, and helping the proliferation of bacteria on the surface of a host. There is currently no very effective method to control infection with osteomyelitis staphylococcus aureus.
Gene knockout, also known as gene targeting, is a novel molecular biology technique, which is a method for changing the genetic characteristics of cells by introducing a constructed targeting vector into target cells through DNA transformation technology, integrating vector DNA into a certain defined site on the genome of the target cells at fixed points or replacing a certain defined fragment on the genome of the target cells by recombination vector DNA sequences and homologous DNA sequences on chromosomes in the target cells, and inactivating or deleting specific genes of the organism. However, the existing bacterial gene knockout methods are more, and bacteria are various, so that the morphological characteristics, the internal structures and the physiological characteristics of the bacteria have larger differences, and the difficulty of the bacterial gene knockout technology is larger; and the gene knockout method for inhibiting the formation of the biological film mediated by the SdrC protein and slowing down the infection of the staphylococcus aureus to a host by a genetic engineering technology is not reported.
In view of the above, it is necessary to study a method for knocking out the SdrC protein gene of Staphylococcus aureus.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for knocking out a staphylococcus aureus SdrC protein gene, which aims to solve the problems in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a method for knocking out a staphylococcus aureus SdrC protein gene, comprising the following steps:
(1) According to the genome sequence of staphylococcus aureus, an upstream primer RN4220-SdrC-up-F and a downstream primer RN4220-SdrC-up-R of an upstream homologous arm sequence of the SdrC protein are designed, and an upstream primer RN4220-SdrC-down-R and a downstream primer RN4220-SdrC-down-F of a downstream homologous arm sequence of the SdrC protein are designed;
(2) Amplifying by adopting an overlap PCR amplification method by taking genome DNA of staphylococcus aureus as a template and using the upstream primer RN4220-SdrC-up-F and the downstream primer RN4220-SdrC-up-R to obtain an upstream homology arm fragment of SdrC protein; amplifying by using the upstream primer RN4220-SdrC-down-R and the downstream primer RN4220-SdrC-down-F to obtain a downstream homology arm fragment of the protein SdrC;
(3) Overlapping PCR amplification is carried out by taking the upstream homology arm segment and the downstream homology arm segment as templates, and the upstream homology arm segment and the downstream homology arm segment are fused to obtain a fusion segment of an upstream homology arm and a downstream homology arm of the SdrC protein;
(4) Amplifying the SdrC homologous arm fragments by using an upstream primer sdrC-pkOR1-F and a downstream primer sdrC-pkOR1-R to construct a knockout vector, preparing a pKOR1 granulating vector frame, and then performing seamless cloning to construct an SdrC recombinant suicide plasmid;
(5) And electrically converting the constructed SdrC recombinant suicide plasmid into a wild strain of staphylococcus aureus, and obtaining the staphylococcus aureus with the SdrC protein gene knocked out by constructing a deletion mutant strain after screening.
Preferably, in step (1), the base sequences of the upstream primer RN4220-sdrc-up-F and the downstream primer RN4220-sdrc-up-R of the upstream homology arm sequence are respectively:
RN4220-sdrc-up-F：ATTCTGGAATATTGAAAAATGCA
RN4220-sdrc-up-R：GTTAAGTTAATCATAAATCAATGTTGTCCCTACTAA
the base sequences of the upstream primer RN4220-sdrc-down-R and the downstream primer RN4220-sdrc-down-F of the downstream homology arm sequence are respectively as follows:
RN4220-sdrc-down-R：TTTCATCATTACTCTTGATAGCA
RN4220-sdrc-down-F：GGGACAACATTGATTTATGATTAACTTAACCAGGTCCA。
preferably, in step (3), the amplification conditions of the overlapping PCR amplification are: 94 ℃ for 5min 2cycle,94 ℃ for 30sec, 50 ℃ for 30sec and 68 ℃ for 1min;30cycle,94℃for 30sec, 50℃for 30sec, 68℃for 1min,10℃hold on.
Preferably, in the step (4), the base sequences of the upstream primer sdrC-pkoR1-F and the downstream primer sdrC-pkoR1-R are respectively:
Sdrc-pkoR1-F：GCCTCGGAACCGGTACCATTCTGGAATATTGAAAAATG
Sdrc-pkoR1-R：GTGAGCGCAACGCAATTTTCATCATTACTCTTGATAG。
preferably, in the step (4), the base sequences of the upstream primer pKOR1-sdrC-F and the downstream primer pKOR1-sdrC-R when preparing the pKOR1 pelletization carrier frame are respectively:
pkoR1-sdrc-F：CAAGAGTAATGATGAAAATTGCGTTGCGCTCACTGCCCG
pkoR1-sdrc-R：TTTCAATATTCCAGAATGGTACCGGTTCCGAGGCTCAA。
preferably, in step (5), the base sequences of the identification primers identified by colony PCR during the screening are respectively:
Sdrc-JD-F:ATGTAATAGCGAATTGAAAAC
Sdrc-JD-R:ATTGGATACCATGCCTTTCCTT。
preferably, in step (5), the method for constructing a deletion mutant is as follows: positive clones were inoculated into 5ml of TSB, cm 10. Mu.g/ml liquid medium, incubated at 30℃and transferred to fresh 5ml of TSB, cm 10. Mu.g/ml, incubated overnight at 43℃and coated with TSA, cm 7.5. Mu.g/ml, incubated at 43℃and picked up and transferred to fresh 5ml of TSB, cm 5. Mu.g/ml, incubated overnight at 43℃and coated with TSA, incubated at 30℃and picked up and transferred to 5ml of TSB at 30℃and bacterial liquid was diluted and applied to ATC, 1. Mu.g/ml plates, grown clones were spotted onto TSA plates and TSA, cm 10. Mu.g/ml plates and non-growing clones on chloramphenicol plates were picked up for identification.
Preferably, when a clone which is not long on a chloramphenicol plate is selected for identification, primers which are different from Sdrc-JD-F and Sdrc-JD-R, sdrc-JD-F and Sdrc-ter-R are adopted for verification; the base sequence of the sdrc-ter-R is as follows: CTTGTTCAAAGCTACCTCTAAC.
In summary, compared with the prior art, the invention has the following advantages:
according to the method for knocking out the staphylococcus aureus SdrC protein gene, the SdrC recombinant suicide plasmid is constructed, the knocking-out vector is constructed, the RN4220 wild strain is electrically transformed from the vector, the deletion mutant strain is constructed, finally, the SdrC amino acid fragment with the same functional annotation is successfully knocked out, the formation of a biological film mediated by the SdrC protein is inhibited, and the infection of the staphylococcus aureus to a host is slowed down.
Drawings
FIG. 1 is a diagram of PCR-verified srrC amplified fragments of the present invention; in the figure, lane M, DL2000 DNA Marker; lane 1:A fragment; lane 2:B fragment.
FIG. 2 is a diagram of PCR-verified overlapping amplified fragments according to the present invention; in the figure, lane M, DL2000 DNA Marker; lane 1, fusion AB fragment.
FIG. 3 is an amplification of the srrC homology arm fragment of the present invention; in the figure, lane M, DL2000 DNA Marker; lane 1 shows the srrC homology arm fragment.
FIG. 4 is a diagram showing the detection of 1% agarose gel electrophoresis of the amplification of the pKOR1 vector frame of the present invention;
FIG. 5 is a graph showing PCR identification results of the RN4220 transformed colonies of the present invention;
FIG. 6 is a graph showing the results of the verification of the present invention using different primers; FIG. a shows the wild strain and the knocked-out strain of the sdrC-JD-F/JD-R, and FIG. b shows the wild strain and the knocked-out strain of the sdrC-JD-F/sdrC-ter-R; wherein 1 refers to a knocked-out strain RN4220 delta sdrc amplification product, and 2 refers to a wild strain RN4220 control;
FIG. 7 is a sequence diagram of mRNA and upstream and downstream genes of the SdrC protein of Staphylococcus aureus of the present invention; in the figure, the target gene is underlined, the primer is marked in the frame, and the gray background is marked that the other gene overlapping region of the annotation needs to be reserved;
FIG. 8 is a gene sequence diagram of the invention after knocking out the SdrC protein of the golden yellow grape.
Detailed Description
The present invention will be further described below.
The method for knocking out the SdrC protein gene in the staphylococcus aureus RN4220 genome provided by the embodiment comprises the following steps:
1. primer design
According to the genome sequence of staphylococcus aureus RN4220, an upstream primer RN4220-SdrC-up-F and a downstream primer RN4220-SdrC-up-R of an upstream homologous arm sequence of the SdrC protein are designed, and an upstream primer RN4220-SdrC-down-R and a downstream primer RN4220-SdrC-down-F of a downstream homologous arm sequence of the SdrC protein are designed;
RN4220-sdrc-up-F：ATTCTGGAATATTGAAAAATGCA
RN4220-sdrc-up-R：GTTAAGTTAATCATAAATCAATGTTGTCCCTACTAA
RN4220-sdrc-down-R：TTTCATCATTACTCTTGATAGCA
RN4220-sdrc-down-F：GGGACAACATTGATTTATGATTAACTTAACCAGGTCCA。
2. overlapping PCR amplified staphylococcus aureus RN4220-SdrC fusion fragment
2.1 amplification of homologous arms upstream and downstream of Gene
Amplifying with RN4220-sdrC-up-F/RN4220-sdrC-up-R to obtain an upstream homology arm A fragment of SdrC;
amplifying with RN4220-sdrC-down-F/RN4220-sdrC-down-R to obtain a downstream homology arm B fragment of the SdrC;
the amplification procedure was as follows:
amplification conditions: 94℃5min 2Cycle (94℃30sec, 55℃30sec, 72℃40 sec), 30Cycle (94℃30sec, 55℃30sec, 72℃40 sec) 10℃hold on.
The amplification results of the PCR-verified sdrC amplified fragments are shown in FIG. 1.
2.2 homologous arm fusion of the upstream and downstream homology arm genes of SDRC Gene
Overlapping PCR amplification is carried out by taking an upstream homology arm A fragment of the SdrC and a downstream homology arm B fragment of the SdrC as templates; the AB fragment was fused.
The amplification procedure was as follows:
amplification conditions: 94℃5min 2cycle (94℃30sec, 50℃30sec, 68℃ 1 min), 30cycle (94℃30sec, 50℃30sec, 68℃ 1 min) 10℃hold on.
The PCR-verified overlapping amplified fragment amplification results are shown in FIG. 2.
3. Construction of srrC recombinant suicide plasmid
3.1 knockout vector construction
The srrc homologous fragment was amplified as follows:
amplification conditions: 94℃5min 32cycle (94℃30sec, 50℃30sec, 68℃40 sec) 10℃hold on;
the base sequences of the upstream primer sdrC-pkoR1-F and the downstream primer sdrC-pkoR1-R are respectively as follows:
Sdrc-pkoR1-F：GCCTCGGAACCGGTACCATTCTGGAATATTGAAAAATG
Sdrc-pkoR1-R：GTGAGCGCAACGCAATTTTCATCATTACTCTTGATAG。
the result of amplification of the srrc homology arm fragment is shown in fig. 3.
3.2 preparation of pkor1 vector frame:
the amplification procedure was as follows:
amplification conditions: 94℃5min 32cycle (94℃30sec, 50℃30sec, 68℃ 3 min) 10℃hold on.
The base sequences of the upstream primer pKOR1-sdrC-F and the downstream primer pKOR1-sdrC-R when preparing the pKOR1 pelletization vector frame were respectively:
pkoR1-sdrc-F：CAAGAGTAATGATGAAAATTGCGTTGCGCTCACTGCCCG
pkoR1-sdrc-R：TTTCAATATTCCAGAATGGTACCGGTTCCGAGGCTCAA。
the results of the pKOR1 vector frame amplification 1% agarose gel electrophoresis are shown in FIG. 4.
3.3 seamless cloning
The amplification procedure was as follows:
the cells were placed on ice for 30min, ecoli DH5alphA competent cells were transformed, amp resistance plates were plated, incubated overnight at 37℃and positive clones were picked and sequenced.
4. Transformation screening
Knock-out vector electrotransformation RN4220
Plates of TSA (Cm 5. Mu.g/ml) were plated and incubated at 30℃and colonies were PCR identified:
identifying a primer:
Sdrc-JD-F:ATGTAATAGCGAATTGAAAAC
Sdrc-JD-R:ATTGGATACCATGCCTTTCCTT
the system comprises: the monoclonal is picked up into 10 mu l of sterile water, and after being evenly mixed, 0.5 mu l is taken as a template according to the following system:
amplification conditions: 94℃for 5min,32 cycles (98℃for 5sec,55℃for 5sec,68℃for 10 sec), 10℃hold on.
6 colonies were selected for colony PCR identification, and the results of colony PCR identification for RN4220 transformation are shown in FIG. 5.
5. Construction of deletion mutant
Positive clones were inoculated into 5ml TSB (Cm 10. Mu.g/ml) liquid medium, incubated at 30℃and transferred to fresh 5ml TSB (Cm 10. Mu.g/ml) 43℃overnight the next day, TSA (Cm 7.5. Mu.g/ml) plates were plated 43℃and selected for transfer to fresh 5ml TSB (Cm 5. Mu.g/ml), 43℃overnight incubation, TSA plates were plated for 30℃and selected for transfer to 5ml TSB 30℃for culture, bacterial liquid was diluted and plated onto ATC (1. Mu.g/ml) plates, grown clones were spotted onto TSA plates and TSA (Cm 10. Mu.g/ml) plates, and colonies not long on chloramphenicol plates were picked for identification. When a long clone on a chloramphenicol flat plate is selected for identification, primers with different Sdrc-JD-F, sdrc-JD-R, sdrc-JD-F and Sdrc-ter-R are adopted for verification together with JD-F1175 bp;
the base sequence of the srrc-ter-R is: CTTGTTCAAAGCTACCTCTAAC.
The result of the srdC-JD-F/JD-R wild bacteria and the result of the knockout bacteria are shown in FIG. 6a,
the srrc-JD-F/srrc-ter-R wild-type bacterium and the knocked-out bacterium are shown in FIG. 6 b.
In the figure, 1 indicates the amplified product of the knockdown strain RN4220 delta srrc, and 2 indicates the wild-type strain RN4220 control.
The product No. 1 in the figure is sequenced, sequencing results prove that the SdrC deletion mutant is successfully constructed, and the results are shown in fig. 7 and 8.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.
Claims (8)
1. A method for knocking out a staphylococcus aureus SdrC protein gene, which is characterized by comprising the following steps:
(1) According to the genome sequence of staphylococcus aureus, an upstream primer RN4220-SdrC-up-F and a downstream primer RN4220-SdrC-up-R of an upstream homologous arm sequence of the SdrC protein are designed, and an upstream primer RN4220-SdrC-down-R and a downstream primer RN4220-SdrC-down-F of a downstream homologous arm sequence of the SdrC protein are designed;
(2) Amplifying by adopting an overlap PCR amplification method by taking genome DNA of staphylococcus aureus as a template and using the upstream primer RN4220-SdrC-up-F and the downstream primer RN4220-SdrC-up-R to obtain an upstream homology arm fragment of SdrC protein; amplifying by using the upstream primer RN4220-SdrC-down-R and the downstream primer RN4220-SdrC-down-F to obtain a downstream homology arm fragment of the protein SdrC;
(3) Overlapping PCR amplification is carried out by taking the upstream homology arm segment and the downstream homology arm segment as templates, and the upstream homology arm segment and the downstream homology arm segment are fused to obtain a fusion segment of an upstream homology arm and a downstream homology arm of the SdrC protein;
(4) Amplifying the SdrC homologous arm fragments by using an upstream primer sdrC-pkOR1-F and a downstream primer sdrC-pkOR1-R to construct a knockout vector, preparing a pKOR1 granulating vector frame, and then performing seamless cloning to construct an SdrC recombinant suicide plasmid;
(5) And electrically converting the constructed SdrC recombinant suicide plasmid into a wild strain of staphylococcus aureus, and obtaining the staphylococcus aureus with the SdrC protein gene knocked out by constructing a deletion mutant strain after screening.
2. The method of claim 1, wherein in the step (1), the base sequences of the upstream primer RN4220-SdrC-up-F and the downstream primer RN4220-SdrC-up-R of the upstream homology arm sequence are respectively:
RN4220-sdrc-up-F：ATTCTGGAATATTGAAAAATGCA
RN4220-sdrc-up-R：GTTAAGTTAATCATAAATCAATGTTGTCCCTACTAA
the base sequences of the upstream primer RN4220-sdrc-down-R and the downstream primer RN4220-sdrc-down-F of the downstream homology arm sequence are respectively as follows:
RN4220-sdrc-down-R：TTTCATCATTACTCTTGATAGCA
RN4220-sdrc-down-F：GGGACAACATTGATTTATGATTAACTTAACCAGGTCCA。
3. the method of claim 1, wherein in step (3), the amplification conditions of the overlap PCR amplification are: 94 ℃ for 5min 2cycle,94 ℃ for 30sec, 50 ℃ for 30sec and 68 ℃ for 1min;30cycle,94℃for 30sec, 50℃for 30sec, 68℃for 1min,10℃hold on.
4. The method of claim 1, wherein in step (4), the base sequences of the upstream primer SdrC-pkoR1-F and the downstream primer SdrC-pkoR1-R are respectively:
Sdrc-pkoR1-F：GCCTCGGAACCGGTACCATTCTGGAATATTGAAAAATG
Sdrc-pkoR1-R：GTGAGCGCAACGCAATTTTCATCATTACTCTTGATAG。
5. the method of claim 1, wherein in step (4), the base sequences of the upstream primer pKOR1-SdrC-F and the downstream primer pKOR1-SdrC-R in preparing pKOR1 pelleting vector frame are respectively:
pkoR1-sdrc-F：CAAGAGTAATGATGAAAATTGCGTTGCGCTCACTGCCCG
pkoR1-sdrc-R：TTTCAATATTCCAGAATGGTACCGGTTCCGAGGCTCAA。
6. the method of claim 1, wherein in step (5), the base sequences of the identification primers for colony PCR identification are respectively:
Sdrc-JD-F:ATGTAATAGCGAATTGAAAAC
Sdrc-JD-R:ATTGGATACCATGCCTTTCCTT。
7. the method for knocking out a staphylococcus aureus SdrC protein gene according to claim 1, wherein in the step (5), the method for constructing a deletion mutant is as follows: positive clones were inoculated into 5ml of TSB, cm 10. Mu.g/ml liquid medium, incubated at 30℃and transferred to fresh 5ml of TSB, cm 10. Mu.g/ml, incubated overnight at 43℃and coated with TSA, cm 7.5. Mu.g/ml, incubated at 43℃and picked up and transferred to fresh 5ml of TSB, cm 5. Mu.g/ml, incubated overnight at 43℃and coated with TSA, incubated at 30℃and picked up and transferred to 5ml of TSB at 30℃and bacterial liquid was diluted and applied to ATC, 1. Mu.g/ml plates, grown clones were spotted onto TSA plates and TSA, cm 10. Mu.g/ml plates and non-growing clones on chloramphenicol plates were picked up for identification.
8. The method for knocking out staphylococcus aureus SdrC protein according to claim 7, wherein when the non-long clone on the chloramphenicol plate is selected for identification, primers different from Sdrc-JD-F and Sdrc-JD-R, sdrC-JD-F and Sdrc-ter-R are adopted for verification; the base sequence of the sdrc-ter-R is as follows: CTTGTTCAAAGCTACCTCTAAC.
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