CN110656119A - Expression vector for observing bacterial protein localization and construction method thereof - Google Patents
Expression vector for observing bacterial protein localization and construction method thereof Download PDFInfo
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Abstract
The invention discloses an expression vector for observing bacterial protein localization and a construction method thereof. Firstly, an expression vector containing an inducible tac promoter and a green fluorescent protein GFP gene is constructed, a target protein gene is inserted into the vector to enable the target protein and the green fluorescent protein GFP to be fused and expressed, then the vector is transferred into bacteria and induced to express, and the spatial positioning of the target protein is determined by observing the distribution condition of green fluorescence in bacterial cells under a fluorescence microscope. The invention can realize the real-time and intuitive detection of the space positioning of the target protein in the living cells, and is convenient and quick.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an expression vector for observing bacterial protein localization and a construction method thereof.
Background
The protein is the final executor of life activity, the subcellular localization of protein is closely related to its function, and the correct localization of protein in cell is the prerequisite guarantee for the highly ordered operation of cell system. The research on the mechanism and the law of protein localization in cells and the prediction of the subcellular localization of proteins have important significance for understanding the properties and the functions of the proteins, the interaction among the proteins and the exploration of the law and the secret of life. The method for detecting the space orientation of the target protein by fusing the target protein and the fluorescent protein and utilizing the fluorescence characteristic of the fluorescent protein is a quick and sensitive method for researching the space orientation of the protein. Green Fluorescent Protein (GFP) emits Green fluorescence under excitation of blue wavelength light, which makes GFP a widely used as a tag protein in biotechnology. The GFP mediated fusion protein labeling technology is a recombinant DNA technology based on a reporter gene, which is started at the end of the 20 th century, and can be applied to detection of gene expression level in cells, in-situ tracing and positioning of proteins in cells and the like.
Bacteria belong to prokaryotes, and there are many cases of intracellular protein localization, such as on membranes, near the ends of bacterial cells, in cytoplasm, in periplasmic space, etc., and these different localizations are closely related to the function of proteins. At present, most of common protein space positioning vectors are designed aiming at eukaryotic cells, and less protein positioning vectors aim at prokaryotic cells.
Disclosure of Invention
In view of the above, the present invention provides an expression vector for observing bacterial protein localization and a construction method thereof, and the expression vector of the present invention not only can visually detect the spatial localization of general proteins in bacteria, but also is suitable for the localization observation of proteins with low expression amount, toxic proteins or proteins expressed at a specific stage of bacterial growth, and is convenient and practical.
The technical scheme of the invention is realized as follows:
in one aspect, the invention provides an expression vector for observing bacterial protein localization, which comprises an inducible expression element, a green fluorescent protein gfp gene, a junction transfer site, a multiple cloning site and a gene encoding a target protein, wherein the nucleotide sequence of the green fluorescent protein gfp gene is shown as SEQ ID No.1, the gene encoding the target protein is inserted into the multiple cloning site, and the target protein comprises a protein expressed only at a specific stage of bacterial growth.
On the basis of the above technical scheme, preferably, the inducible expression element consists of a tac promoter and a lacI operator, and the nucleotide sequence of the inducible expression element is shown as SEQ ID No. 2.
On the basis of the technical scheme, the multiple cloning sites are positioned between the inducible expression element and the GFP gene, namely, the target protein gene is positioned between the inducible expression element and the GFP gene, so that the fusion expression of the target protein and the GFP protein is realized.
In a second aspect, the present invention provides a method for constructing an expression vector for observing bacterial protein localization, comprising the following steps:
s1, reverse amplification is carried out by taking the pBBR1MCS-5 plasmid as a template to obtain a DNA fragment except the lacZ gene on the pBBR1 MCS-5;
s2, amplifying by taking pVLT33 plasmid as a template to obtain a lacI gene + tac promoter;
s3, carrying out enzyme digestion on the DNA fragment obtained in the step S1 and the lacI gene + tac promoter obtained in the step S2 by using restriction enzymes, and then connecting to obtain an expression vector pBBRlacITac;
s4, taking pBBR1-gfp plasmid as a template, and amplifying to obtain gfp gene;
s5, carrying out enzyme digestion on the expression vector pBBRlacetac and gfp genes by using restriction enzymes, and then connecting to obtain an expression vector pBBRlacetac-gfp;
s6, amplifying to obtain a gene x for coding a target protein by taking the total DNA of the bacterial genome as a template;
s7, connecting the gene x for coding the target protein to the expression vector pBBRlacITac-gfp to obtain the expression vector pBBRlacITac-x-gfp.
On the basis of the technical scheme, preferably, the complete nucleotide sequence of the expression vector pBBRlacITac-gfp is shown as SEQ ID No. 3.
Based on the above technical solutions, it is preferable that the restriction enzymes in step S3 are XhoI and NdeI, and the restriction enzymes in step S5 are XhoI and HindIII.
On the basis of the technical scheme, the sequence of the gene x for coding the target protein does not contain a translation stop codon, and the translation stop codon is introduced at the tail end of the gfp gene when the gfp gene is amplified.
In a third aspect, the invention also provides the use of an expression vector for observing bacterial protein localization in the detection of bacterial protein spatial localization, which comprises introducing the expression vector into bacteria to induce expression, and then observing the distribution of green fluorescence in bacterial cells under a fluorescence microscope to determine the spatial localization of a target protein.
Compared with the prior art, the expression vector for observing bacterial protein localization and the construction method thereof have the following beneficial effects:
1. the vector pBBRlacITac-gfp has an inducible tac promoter, and can be added with inducers with different concentrations under different time/conditions aiming at different target proteins, so that the time and the yield of protein expression are controlled conveniently, and the vector pBBRlacITac-gfp is suitable for positioning observation of proteins with low expression quantity, toxic proteins or proteins expressed at a specific growth stage of bacteria;
2. the vector takes pBBR1MCS-5 as a framework, is suitable for various bacteria because of belonging to a wide host vector, and simultaneously has mob genes, so that not only can the plasmid be introduced into a target strain by utilizing heat shock transformation and electric transformation, but also the plasmid can be introduced into the target strain by conjugal transfer;
3. among the multiple cloning sites are 7 commonly used restriction endonuclease sites (EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, HindIII), which are highly inclusive and facilitate the insertion of various genes into the vector.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram showing the construction of the expression vector pBBRlaceitac-gfp of example 1.
FIG. 2 is a photograph under a fluorescent microscope of Pseudomonas putida KT2440 carrying pBBRlacITac-gfp vector.
FIG. 3 is a map of the expression vector pBBRlacITac-motA-gfp of example 2.
FIG. 4 is a photograph under a fluorescent microscope of Pseudomonas putida KT2440 carrying pBBRlacITac-motA-gfp vector of example 2.
FIG. 5 is a map of the expression vector pBBRlacITac-katG-gfp of example 3.
FIG. 6 is a photograph under a fluorescent microscope of E.coli K-12 carrying the pBBRlacITac-katG-gfp vector of example 3.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The experimental procedures in the following examples were carried out by a conventional method unless otherwise specified, and the experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The experimental E.coli strains DH5 alpha, E.coli K-12 and Pseudomonas putida KT2440 were purchased from Beijing Quanjin and grown in conventional LB medium, and selected for growth at antibiotic concentrations: gentamicin Gm 40. mu.g/mL. The original plasmids pBBR1MCS-5, pVLT33 and pBBR1-gfp used in the experiments were purchased from biotech companies and are commonly available on the market. The cloning procedure for the vector is described in the molecular cloning protocols.
Example 1 construction of expression vector
S1, construction of expression vector pBBRlacITac-gfp and fluorescence detection
FIG. 1 shows a schematic diagram of the construction of expression vector pBBRlacITac-gfp, which reverse-amplifies the fragment of pBBR1MCS-5 except lacZ using primer pair P1/P2 and pBBR1MCS-5 plasmid as template; amplifying lacI gene and tac promoter by using a primer pair P3/P4 and pVLT33 plasmid as a template; the two PCR products are cut by NdeI and XhoI enzyme, are connected by T4DNA ligase, and are subjected to sequencing verification to obtain a vector pBBRlacITac. Amplifying a gfp gene by using a primer pair P5/P6 and pBBR1-gfp plasmid as a template; and (3) digesting the gfp gene by using restriction enzyme XhoI + HindIII, then connecting the digested gfp gene to a pBBRlaceitac vector digested by the same enzyme by using T4DNA ligase, and verifying sequencing to determine that the sequence of the gfp gene is correct, thereby finally obtaining the expression vector pBBRlaceitac-gfp. In the expression vector pBBRlacITac-gfp shown in FIG. 1, tac promoter and lacI constitute inducible expression elements; gentamicin-R is a gentamicin resistance gene for screening; the mob gene allows plasmids to be transferred in different strains by conjugal transfer; multiple cloning sites are arranged between the tac promoter and the gfp gene, 7 common restriction enzyme cutting sites exist, and the target gene can be connected into an expression vector pBBRlacITac-gfp through the cutting sites to realize fusion expression.
The pBBRlacITac-gfp vector was introduced into Pseudomonas putida KT2440 by electrotransformation, cultured at 28 ℃ for 12 hours, then induced with 0.5mM IPTG for 3 hours, and then fluorescence was observed under a fluorescence microscope. As a result, as shown in FIG. 2, the cells fluoresced and the fluorescence was uniformly distributed throughout the cells, indicating that the GFP protein was uniformly distributed in the cells without the target gene inserted therein and that no specific localization was observed.
S2 construction of expression vector pBBRlacITac-x-gfp
The coding gene X of the target protein X is connected between the tac promoter of the pBBRlacITac-gfp vector and the gfp gene by using a multiple cloning site to form a fusion expression vector pBBRlacITac-X-gfp of the target protein X.
Example 2 testing of the feasibility of the invention Using the MotA protein as the protein of interest
S1 construction of expression vector pBBRlacITac-motA-gfp
The map of the expression vector pBBRlacITac-motA-gfp is shown in FIG. 3. Pseudomonas putida KT2440 is a single-ended Habrothrix flagellatus. MotA is a KT2440 flagellar matrix component protein, localized at the bacterial endpoints. The pBBRlacITac-gfp vector is used for detecting the spatial localization of MotA in KT2440 and verifying whether the vector has expected functions. The total DNA of a pseudomonas putida KT2440 genome is used as a template, a primer pair P7/P8 is utilized to amplify the motA gene, then EcoRI and BamHI are used to be connected into a pBBRlacetac-gfp vector, sequencing verification is carried out, the motA gene is confirmed to be inserted into the pBBRlacetac-gfp vector, and finally the expression vector pBBRlacetac-motA-gfp is obtained. The PCR-amplified motA coding region has no translation termination codon, so that fusion expression with the downstream gfp gene can be achieved after ligation to a vector.
S2 visual detection of space localization of MotA protein
The pBBRlacITac-motA-gfp vector was introduced into Pseudomonas putida KT2440 by electrotransformation, cultured at 28 ℃ for 12 hours, then induced with IPTG for 3 hours, and then fluorescence was observed under a fluorescence microscope. As a result, as shown in FIG. 4, the fluorescence of the cells was concentrated at one end of the cells, indicating that MotA was localized at one end of the bacteria, consistent with the results reported previously.
Example 3 testing of feasibility of the invention Using KatG protein as the protein of interest
S1 construction of expression vector pBBRlacITac-katG-gfp
The map of the expression vector pBBRlacITac-katG-gfp is shown in FIG. 5. KatG is a catalase in Escherichia coli, and can hydrolyze hydrogen peroxide in cells to relieve oxidative stress crisis. The expression of KatG is induced by hydrogen peroxide. If hydrogen peroxide or other oxidative stress substances are not added into the culture medium, the KatG can only be expressed in a small amount at the later stage of the growth stage of the escherichia coli, and the spatial localization of the KatG is difficult to observe. In the invention, the spatial localization of KatG is observed in the logarithmic growth phase of escherichia coli under the condition of not adding hydrogen peroxide. And (3) amplifying the katG gene by using Escherichia coli K-12 genome DNA as a template and using a primer pair P9/P10, connecting the katG gene to pBBRlacITac-gfp by using XbaI and HindIII enzyme cutting sites, and verifying the sequencing to determine that the katG sequence is correct to obtain a vector pBBRlacITac-katG-gfp. The katG coding region amplified by PCR does not have a translation termination codon, so that the katG coding region can be connected to a vector to realize fusion expression with a downstream gfp gene.
S2, visual detection of KatG protein space orientation
The pBBRlacITac-katG-gfp vector was introduced into E.coli K-12 by electroporation, cultured at 37 ℃ for 12 hours, then induced with IPTG for 3 hours, and then fluorescence was observed under a fluorescence microscope. The results are shown in FIG. 6, where the intensity of the fluorescence of the cells was uniform and the fluorescence was distributed uniformly throughout the cells, indicating that KatG was localized in the cytoplasm of E.coli.
TABLE 1 primer sequences used in the present invention
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Wuhan Boohot Biotechnology Ltd
<120> expression vector for observing bacterial protein localization and construction method thereof
<130> 2019-9-24
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ttaattttct ctggggaaaa gcctccggcc tgcggcctgc gcgcttcgct tgccggttgg 1260
acaccaagtg gaaggcgggt caaggctcgc gcagcgaccg cgcagcggct tggccttgac 1320
gcgcctggaa cgacccaagc ctatgcgagt gggggcagtc gaaggcgaag cccgcccgcc 1380
tgccccccga gcctcacggc ggcgagtgcg ggggttccaa gggggcagcg ccaccttggg 1440
caaggccgaa ggccgcgcag tcgatcaaca agccccggag gggccacttt ttgccggagg 1500
gggagccgcg ccgaaggcgt gggggaaccc cgcaggggtg cccttctttg ggcaccaaag 1560
aactagatat agggcgaaat gcgaaagact taaaaatcaa caacttaaaa aaggggggta 1620
cgcaacagct cattgcggca ccccccgcaa tagctcattg cgtaggttaa agaaaatctg 1680
taattgactg ccacttttac gcaacgcata attgttgtcg cgctgccgaa aagttgcagc 1740
tgattgcgca tggtgccgca accgtgcggc accctaccgc atggagataa gcatggccac 1800
gcagtccaga gaaatcggca ttcaagccaa gaacaagccc ggtcactggg tgcaaacgga 1860
acgcaaagcg catgaggcgt gggccgggct tattgcgagg aaacccacgg cggcaatgct 1920
gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc gtggtggtca gccagaagac 1980
actttccaag ctcatcggac gttctttgcg gacggtccaa tacgcagtca aggacttggt 2040
ggccgagcgc tggatctccg tcgtgaagct caacggcccc ggcaccgtgt cggcctacgt 2100
ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag ttgcgcctgt cggtgttcag 2160
tgccgccgtg gtggttgatc acgacgacca ggacgaatcg ctgttggggc atggcgacct 2220
gcgccgcatc ccgaccctgt atccgggcga gcagcaacta ccgaccggcc ccggcgagga 2280
gccgcccagc cagcccggca ttccgggcat ggaaccagac ctgccagcct tgaccgaaac 2340
ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg cccgatgagc cgtgttttct 2400
ggacgatggc gagccgttgg agccgccgac acgggtcacg ctgccgcgcc ggtagcactt 2460
gggttgcgca gcaacccgta agtgcgctgt tccagactat cggctgtagc cgcctcgccg 2520
ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga 2580
cctgaatgga agccggcggc acctcgctaa cggattcacc gtttttatca ggctctggga 2640
ggcagaataa atgatcatat cgtcaattat tacctccacg gggagagcct gagcaaactg 2700
gcctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa 2760
ccagcaatag acataagcgg ctatttaacg accctgccct gaacctcgag ctatttgtat 2820
agttcatcca tgccatgtgt aatcccagca gctgttacaa actcaagaag gaccatgtgg 2880
tctctctttt cgttgggatc tttcgaaagg gcagattgtg tggacaggta atggttgtct 2940
ggtaaaagga cagggccatc gccaattgga gtattttgtt gataatggtc tgctagttga 3000
acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc attcttttgt 3060
ttgtctgcca tgatgtatac attgtgtgag ttatagttgt attccaattt gtgtccaaga 3120
atgtttccat cttctttaaa atcaatacct tttaactcga ttctattaac aagggtatca 3180
ccttcaaact tgacttcagc acgtgtcttg tagttcccgt catctttgaa aaatatagtt 3240
ctttcctgta cataaccttc gggcatggca ctcttgaaaa agtcatgctg tttcatatga 3300
tctgggtatc tcgcaaagca ttgaacacca taaccgaaag tagtgacaag tgttggccat 3360
ggaacaggta gttttccagt agtgcaaata aatttaaggg taagttttcc gtatgttgca 3420
tcaccttcac cctctccact gacagaaaat ttgtgcccat taacatcacc atctaattca 3480
acaagaattg ggacaactcc agtgaaaagt tcttctcctt tactcataag cttgcatgcc 3540
tgcaggtcga ctctagagga tccccgggta ccgagctcga attctgtttc ctgtgtgaaa 3600
ttgttatccg ctcacaattc cacacattat acgagccgat gattaattgt caacagctca 3660
tttcagaata tttgccagaa ccgttatgat gtcggcgcaa aaaacattat ccagaacggg 3720
agtgcgcctt gagcgacacg aattatgcag tgatttacga cctgcacagc cataccacag 3780
cttccgatgg ctgcctgacg ccagaagcat tggtgcaccg tgcagtcgat aagcccggat 3840
cagcttgcaa ttcgcgcgcg aaggcgaagc ggcatttacg ttgacaccat cgaatggtgc 3900
aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat 3960
gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt 4020
tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg 4080
gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag 4140
tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc 4200
gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa 4260
cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt 4320
gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc 4380
actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt 4440
ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag 4500
caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc 4560
tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg 4620
agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact 4680
gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc 4740
gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca 4800
tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccacg 4860
tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg 4920
tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg 4980
cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 5040
gagcgcaacg caattaatgt aagttagcgc gaattgcaag ctggccgacg cgctgggcta 5100
cgtcttgctg gccatatggt tcggggcatc agcaccttgt cgccttgcgt ataatatttg 5160
cccatggacg cacaccgtgg aaacggatga aggcacgaac ccagttgaca taagcctgtt 5220
cggttcgtaa actgtaatgc aagtagcgta tgcgctcacg caactggtcc agaaccttga 5280
ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt 5340
ttgtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt 5400
tgatgttatg gagcagcaac gatgttacgc agcagcaacg atgttacgca gcagggcagt 5460
cgccctaaaa caaagttagg tggctcaagt atgggcatca ttcgcacatg taggctcggc 5520
cctgaccaag tcaaatccat gcgggctgct cttgatcttt tcggtcgtga gttcggagac 5580
gtagccacct actcccaaca tcagccggac tccgattacc tcgggaactt gctccgtagt 5640
aagacattca tcgcgcttgc tgccttcgac caagaagcgg ttgttggcgc tctcgcggct 5700
tacgttctgc ccaggtttga gcagccgcgt agtgagatct atatctatga tctcgcagtc 5760
tccggcgagc accggaggca gggcattgcc accgcgctca tcaatctcct caagcatgag 5820
gccaacgcgc ttggtgctta tgtgatctac gtgcaagcag attacggtga cgatcccgca 5880
gtggctctct atacaaagtt gggcatacgg gaagaagtga tgcactttga tatcgaccca 5940
agtaccgcca cctaacaatt cgttcaagcc gagatcggct tcccggccgc ggagttgttc 6000
ggtaaattgt cacaacgccg ccaggtggca cttttcgggg aaatgtgcgc gcccgcgttc 6060
ctgctggcgc tgggcctgtt tctggcgctg gacttcccgc tgttccgtca gcagcttttc 6120
gcccacggcc ttgatgatcg cggcggcctt ggcctgcata tcccgattca acggccccag 6180
ggcgtccaga acgggcttca ggcgctcccg aaggt 6215
<210> 4
<211> 31
<212> DNA
<213> (Artificial sequence)
<400> 4
ggaattccat atgggcatca gcaccttgtc g 31
<210> 5
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 5
ccgctcgagg ttcagggcag ggtcgtt 27
<210> 6
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 6
ggaattccat atgccgaacg ccagcaagac 30
<210> 7
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 7
ccgctcgaga tccgccaaaa cagccaa 27
<210> 8
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 8
cccaagctta tgagtaaagg agaagaa 27
<210> 9
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 9
ccgctcgagc tatttgtata gttcatc 27
<210> 10
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 10
ccggaattca tggctaaaat tatcggc 27
<210> 11
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 11
cgcggatccg cgaccgcgaa ccgcttg 27
<210> 12
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 12
tgctctagaa tgagcacgtc agacgat 27
<210> 13
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 13
cccaagcttc agcaggtcga aacggtc 27
Claims (8)
1. An expression vector for observing bacterial protein localization, which comprises an inducible expression element, a green fluorescent protein gfp gene, a junction transfer site, a multiple cloning site and a gene coding a target protein, and is characterized in that: the nucleotide sequence of the green fluorescent protein gfp gene is shown as SEQ ID No.1, the gene coding the target protein is inserted into the multiple cloning site, and the target protein comprises a protein expressed only at a specific growth stage of bacteria.
2. The expression vector for observing bacterial protein localization according to claim 1, wherein: the inducible expression element consists of a tac promoter and a lacI operator, and the nucleotide sequence of the inducible expression element is shown as SEQ ID No. 2.
3. The expression vector for observing bacterial protein localization according to claim 1, wherein: the multiple cloning site is located between the inducible expression element and the gfp gene.
4. A construction method of an expression vector for observing bacterial protein localization is characterized by comprising the following steps:
s1, reverse amplification is carried out by taking the pBBR1MCS-5 plasmid as a template to obtain a DNA fragment except the lacZ gene on the pBBR1 MCS-5;
s2, amplifying by taking pVLT33 plasmid as a template to obtain a lacI gene + tac promoter;
s3, carrying out enzyme digestion on the DNA fragment obtained in the step S1 and the lacI gene + tac promoter obtained in the step S2 by using restriction enzymes, and then connecting to obtain an expression vector pBBRlacITac;
s4, taking pBBR1-gfp plasmid as a template, and amplifying to obtain gfp gene;
s5, carrying out enzyme digestion on the expression vector pBBRlacetac and gfp genes by using restriction enzymes, and then connecting to obtain an expression vector pBBRlacetac-gfp;
s6, amplifying to obtain a gene x for coding a target protein by taking the total DNA of the bacterial genome as a template;
s7, connecting the gene x for coding the target protein to the expression vector pBBRlacITac-gfp to obtain the expression vector pBBRlacITac-x-gfp.
5. The method of claim 4 for constructing an expression vector for observing bacterial protein localization, wherein: the complete nucleotide sequence of the expression vector pBBRlacITac-gfp is shown in SEQ ID No. 3.
6. The method of claim 4 for constructing an expression vector for observing bacterial protein localization, wherein: the restriction enzymes in step S3 were XhoI and NdeI, and the restriction enzymes in step S5 were XhoI and HindIII.
7. The method of claim 4 for constructing an expression vector for observing bacterial protein localization, wherein: the x sequence of the gene for coding the target protein does not contain a translation stop codon, and the translation stop codon is introduced at the tail end of the gfp gene when the gfp gene is amplified.
8. Use of an expression vector according to claim 1 for observing the localization of a bacterial protein in an assay for the spatial localization of a bacterial protein, characterized in that: the expression vector is introduced into bacteria to induce expression, and then the distribution of green fluorescence in bacterial cells is observed under a fluorescence microscope to determine the spatial localization of the target protein.
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| CN113444739A (en) * | 2021-06-20 | 2021-09-28 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Construction of broad-host plasmid for inducible expression of green fluorescent protein and application of broad-host plasmid in fluorescent tracing |
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