CN113046350B - Erythromycin induced promoter and application thereof - Google Patents
Erythromycin induced promoter and application thereof Download PDFInfo
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Abstract
The invention discloses an erythromycin induced promoter and application thereof. The nucleotide sequence of the specific promoter provided by the invention is shown as SEQ ID NO.1 sequence in a sequence table. Experiments prove that the specific promoter provided by the invention can start the expression of a target gene (such as a green fluorescent protein coding gene) when erythromycin is induced, and the specific promoter is an erythromycin-induced promoter. The invention has important application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an erythromycin inducible promoter and application thereof.
Background
A promoter is a DNA sequence located upstream of a structural gene and has transcription initiation specificity, to which RNA polymerase binds and initiates transcription of the gene. The-10 region (TATA region) and the-35 region (TTGACA region) of a prokaryotic promoter are the binding sites for RNA polymerase to the promoter. Compared with a constitutive promoter, the inducible promoter can realize the controllable expression of a downstream target gene by adding an inducer or changing culture conditions and the like, so that the inducible promoter is widely applied to the field of genetic engineering. Escherichia coli has the advantages of clear genetic background (simple gene operation), short growth period, high-density fermentation realization and the like, and can be widely used as a prokaryotic expression host; similarly, klebsiella pneumoniae belonging to the family Enterobacteriaceae has high genetic background homology with Escherichia coli and high propagation speed, and is often used as a prokaryotic expression host. At present, the inducible promoters such as isopropyl-beta-D-thiogalactoside (IPTG), tetracycline or high temperature and the like are widely applied to prokaryotic expression of target proteins, and have advantages and disadvantages.
Enhanced Green Fluorescent Protein (EGFP) is a mutant of Green Fluorescent Protein (GFP) and consists of 238 amino acids with a molecular weight of about 27KDa. The fluorescence intensity of EGFP is more than 6 times larger than that of GFP, and the EGFP has the advantages of small toxic and side effects on host cells, simple and convenient detection method, no need of adding substrates and the like, so the EGFP is widely applied to the field of biological research as a reporter gene.
Disclosure of Invention
The invention aims to provide an erythromycin inducible promoter, and through comparison of NCBI databases and literature research, although the DNA sequence is found in genomes or plasmids such as acinetobacter baumannii, klebsiella pneumoniae, salmonella, pseudomonas aeruginosa, escherichia coli and the like, the sequence is not found and reported to be an erythromycin inducible promoter, and can respond to erythromycin and further start expression of downstream genes. The erythromycin induced promoter can effectively supplement and enrich the current promoter module library, and has important potential application. The expression of a target gene (taking EGFP as an example) is promoted by the erythromycin, so that the target protein can be expressed by taking enterobacteriaceae as a host in the field of genetic engineering, and the erythromycin residue in an environmental medium can be detected in the field of environmental protection.
Technical scheme of the invention
An erythromycin inducible promoter, in particular to a specific DNA molecule shown in a sequence table SEQ ID NO.1 sequence; the invention also provides an expression cassette containing the specific DNA molecule. The expression cassette comprises a promoter region, a transcription initiation region, a gene region of interest, a transcription termination region, and optionally a translation termination region, comprised of the specific DNA molecule.
The invention also provides a recombinant plasmid containing the specific DNA molecule. The recombinant plasmid can be obtained by inserting the specific DNA molecule into a starting plasmid. The recombinant plasmid may comprise an expression cassette for the specific DNA molecule described above. For example, the recombinant plasmids may be specifically the recombinant plasmids pUC18-P _ EGFP and pUC18T-P _ EGFP mentioned in the examples.
The invention also provides a transgenic cell strain containing the specific DNA molecule. For example, transgenic cell lines containing the above specific DNA molecules can be E.coli DH 5. Alpha./pUC 18-P _ EGFP and K.pneumoniae ATCC13883/pUC18T-P _ EGFP as mentioned in the examples. The transgenic cell lines listed above are not limiting. Any transgenic cell line containing the specific DNA molecule described above can be used in the present invention.
The invention also provides the application of the specific DNA molecule, the expression cassette or the recombinant plasmid in starting the expression of target genes.
The application method for starting the expression of the target gene can specifically comprise the following steps:
1. the specific DNA molecule is used as a promoter or an erythromycin-induced promoter and is inserted into the upstream of any target gene or enhancer to start the expression of the target gene;
2. inserting a target gene into the downstream of the specific DNA molecule in the expression cassette, and starting the expression of the target gene by the specific DNA molecule;
3. inserting the target gene into the downstream of the specific DNA molecule in the recombinant plasmid, and starting the expression of the target gene by the specific DNA molecule.
The expression of the target gene may be specifically the expression of the target gene upon erythromycin induction.
The specific DNA molecule can be used as a promoter (particularly an erythromycin-induced promoter) and can express a target gene in prokaryotes (such as E.coli DH5 alpha and K.pneumoconiae ATCC 13883).
The above-mentioned target gene may be the EGFP-encoding gene mentioned in the examples. The EGFP coding gene is shown as a sequence of SEQ ID NO.2 in a sequence table. The amino acid sequence of the EGFP is shown as the sequence of SEQ ID NO.3 in the sequence table.
The inducer of the specific promoter is erythromycin. In one embodiment of the invention (E.coli DH 5. Alpha./pUC 18-P _ EGFP), the optimal final concentration of erythromycin to induce initiation of expression of the gene of interest is 15-35. Mu.g/mL; in another embodiment of the invention (K.pneumoconiae ATCC13883/pUC18T-P _ EGFP), the optimal final concentration of erythromycin to induce initiation of expression of the gene of interest is 5-10. Mu.g/mL.
The invention has the advantages and beneficial effects that:
experiments prove that the specific DNA molecule provided by the invention can start the expression of a target gene (such as an EGFP coding gene) when erythromycin is induced, and the specific DNA molecule is an erythromycin-induced promoter. The invention has important application value.
Drawings
FIG. 1 shows the construction of recombinant plasmids pUC18-P _ EGFP (A) and pUC18T-P _ EGFP (B) containing an erythromycin-inducible promoter controlling the EGFP gene.
Fig. 2 different concentrations of erythromycin induction initiate EGFP expression in e.
FIG. 3 different concentrations of erythromycin induction initiate the expression of EGFP in K.pneumoniae ATCC13883/pUC18T-P _ EGFP.
Detailed Description
The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1:
recombinant plasmids pUC18-P _ EGFP and pUC18T-P _ EGFP (shown in figure 1) containing erythromycin inducible promoter to control EGFP gene were constructed.
(1) Earlier stage screening finds that erythromycin can stimulate the up-regulated expression of drug-resistant genes msrE (and mphE) of Klebsiella pneumoniae 1517 strain (the msrE and the mphE are positioned on an operon and controlled by the same promoter region);
(2) Using genome DNA of Klebsiella pneumoniae 1517 strain as template, and primer
F1:5'-ATTGTATGAATGGAAATTTTGTGGG-3' and
5'-TTTTATTTACTCCTGTTTAGCTCTTGAAAT-3' PCR amplification is carried out to obtain a promoter region sequence F1/R1 (DNA molecule shown in a sequence table SEQ ID NO. 1) of the drug-resistant gene msrE (and mphE); and taking the fragment F1/R1 as a template, and taking the fragment P1:
5'-ATTTCCAACTCCTCCAGGCACATGCGTAAATTGTATGAATGGAAATTTTGTGGGGATCC-3' and
P3:
5'-GTGAACAGCTCCTCGCCCTTGCTCACCATTTTTATTTACTCCTGTTTAGCTCTTGAAAT-3' is used as primer to amplify to obtain P1/P3 fragment;
(3) Plasmid pET28a-EGFP as a template and F2:5'-ATGGTGAGCAAGGGCGAGGAG-3'
And R2:5'-TTACTTGTACAGCTCGTCCATGCCG-3' as a primer, and obtaining an EGFP gene fragment F2/R2 (a DNA molecule shown by a sequence table SEQ ID NO. 2) through PCR amplification; then taking the fragment F2/R2 as a template, and taking P2:5'-ATTTCAAGAGCTAAACAGGAGTAAATAAAAATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3' and P4:5'-TCGACTAGGTACTTACTTGTACAGCTCGTCCATGCCG-3' as primers for amplification to obtain a P2/P4 fragment;
(4) Amplifying by using the fragments P1/P3 obtained in the step (2) and the fragments P2/P4 obtained in the step (3) as templates by using primers F1 and R2 (the same as the above) to obtain a sequence fragment F1/R2 (a DNA molecule shown by a sequence in SEQ ID NO.4 of a sequence table) after fusion of a promoter region of the drug-resistant gene msrE (and mphE) and the EGFP gene;
(5) Connecting the fragment F1/R2 with a Vector pClone007 Versatile Simple Vector to obtain pClone007-P _ EGFP;
(6) Plasmid pClone007-P _ EGFP is used as a template and a primer is used
L1:5'-CAAGCTTGCATGCCTGCAGGTCGACTTACTTGTACAGCTCGTCCATGCCG-3' and
L2:
5'-AGCTCACTCATTAGGCACCCCAGGCATTGTATGAATGGAAATTTTGTGGGGATCC-3' to obtain L1/L2 fragment by amplification (P _ EGFP sequence is amplified and homology arms are added for ligation);
(7) Amplifying a vector framework sequence fragment L3/L4 by using a plasmid pUC18 as a template and L3:5'-GTCGACCTGCAGGCATGC-3' and L4:5'-GCCTGGGGTGCCTAATGAGT-3' as primers;
(8) By using
The recombinase in the Uni Seamless Cloning and analysis Kit is used for homologous recombination and connection of L1/L2 and L3/L4 fragments, and E.coli DH5 alpha is transformed, so as to obtain E.coli DH5 alpha/pUC 18-P _ EGFP.
(9) Taking plasmid pUC18-P _ EGFP as a template, and
l5:5'-CTGTCAGACCAAGTTTACTCATATATACTTT-3' and
l6:5'-ACTCTTCCTTTTTCAATATTATTGAAGC-3' is used as a primer for PCR amplification to obtain a pUC18-P _ EGFP framework fragment L5/L6;
(10) Using plasmid pME6032 as a template, and
l7:5'-TCAATAATATTGAAAAAGGAAGAGTATGAAACCCAACATACCCCTGATCG-3' and
l8, 5'-TATATGAGTAAACTTGGTCTGACAGTCAGCGATCGGCTCGTTGCC-3' is used as a primer for PCR amplification to obtain a tetA gene (DNA molecule shown in a sequence table SEQ ID NO. 5) fragment L7/L8;
(11) By using
Recombinase in the Uni Seamless Cloning and analysis Kit homologously recombinantly ligate the L5/L6 and L7/L8 fragments to transform K.pneumoniae ATCC13883, yielding K.pneumoniae ATCC13883/pUC18T-P _ EGFP.
Through sequencing identification, the recombinant plasmids pUC18-P _ EGFP and pUC18T-P _ EGFP both contain a promoter region of a drug-resistant gene msrE (and mphE) and a sequence fragment F1/R2 (a DNA molecule shown by a sequence in SEQ ID NO.4 of a sequence table) after fusion of the EGFP gene, and the pUC18T-P _ EGFP contains a tetA gene (a DNA molecule shown by a sequence in SEQ ID NO.5 of the sequence table).
Example 2:
expression of EGFP in E.coli DH 5. Alpha./pUC 18-P _ EGFP was initiated by erythromycin induction
(1) Inoculating a small amount of E.coli DH5 alpha/pUC 18-P _ EGFP bacterial solution into 4mL MH (B) culture medium (containing 60 mu g/mL ampicillin), and culturing overnight at 37 ℃ and 200 rpm;
(2) The overnight-cultured strain solution (300. Mu.L) was transferred to 30 mM MHL (B) medium (containing 60. Mu.g/mL ampicillin (12 replicates each) and cultured at 37 ℃ and 200rpm until the early logarithmic phase (OD) 600 About 0.4-0.6);
(3) Adding erythromycin with different concentrations into 12 parallel cells to make their final concentrations respectively 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55 μ g/mL, culturing at 37 deg.C and 200rpm for 4 hr to induce EGFP expression;
(4) After induction expression, respectively using a 50mL centrifuge tube to centrifugally collect 30mL bacterial liquid at low temperature, discarding supernatant, and adding 2mL 20mM PBS buffer solution (pH 7.2-7.4) to resuspend the thalli;
(5) Transferring the bacterial liquid to a 2mL centrifuge tube, centrifuging at low temperature again, discarding supernatant, and adding 1mL of 20mM PBS buffer solution (pH 7.2-7.4) to resuspend the thalli;
(6) Carrying out ultrasonic bacteria breaking (ultrasonic for 15min, ultrasonic for 2sec, intermittent for 2sec, output power 68W), centrifuging, taking a supernatant of a broken bacteria liquid, and detecting fluorescence intensity (EGFP) by using a fluorescence spectrophotometer, wherein the excitation wavelength is set to be 485nm, and the emission wavelength is set to be 528nm.
As shown in fig. 2, erythromycin as an inducer was able to initiate EGFP expression in e.coli DH5 α/pUC18-P _ EGFP, with an optimal final concentration of 15-35 μ g/mL for erythromycin induction to initiate expression of the gene of interest.
Example 3
Erythromycin induction initiates expression of EGFP in K.pneumoconiae ATCCC 13883/pUC18T-P _ EGFP
(1) Inoculating a small amount of K.pneumoconiae ATCCC 13883/pUC18T-P _ EGFP bacterial solution into 4mL MH (B) culture medium (containing 50 ug/mL tetracycline), and culturing at 37 deg.C and 200rpm overnight;
(2) The overnight-cultured strain solution (300. Mu.L) was transferred to 30 mM MHL (B) medium (containing 50. Mu.g/mL tetracycline, 7 aliquots were added in parallel), and the culture was continued at 37 ℃ and 200rpm until the early logarithmic phase (OD) 600 About 0.4-0.6);
(3) Adding erythromycin with different concentrations into 7 parallel cells to make their final concentrations respectively 0, 5, 10, 15, 20, 25 and 30 μ g/mL, culturing at 37 deg.C and 200rpm for 4 hr, and inducing expression of EGFP;
(4) After induction expression, respectively using a 50mL centrifuge tube to centrifugally collect 30mL bacterial liquid at low temperature, discarding supernatant, and adding 2mL 20mM PBS buffer solution (pH 7.2-7.4) to resuspend the thalli;
(5) Transferring the bacterial liquid to a 2mL centrifuge tube, centrifuging at low temperature again, discarding supernatant, and adding 1mL of 20mM PBS buffer solution (pH 7.2-7.4) to resuspend the thalli;
(6) Carrying out ultrasonic bacteria breaking (ultrasonic for 15min, ultrasonic for 2sec, intermittent for 2sec, output power 68W), centrifuging, taking a supernatant of a broken bacteria liquid, and detecting fluorescence intensity (EGFP) by using a fluorescence spectrophotometer, wherein the excitation wavelength is set to be 485nm, and the emission wavelength is set to be 528nm.
As shown in fig. 3, erythromycin as an inducer was able to initiate EGFP expression in k.pneumaniaatcc 13883/pUC18T-P _ EGFP, and the optimal final concentration of erythromycin induced initiation of expression of the gene of interest was 5-10 μ g/mL.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
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<120> erythromycin induced promoter and application thereof
<141> 2021-03-20
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atgctcggaa tgattgccga cggcacaggc tacatcctgc ttgccttcgc gacacgggga 900
tggatggcgt tcccgatcat ggtcctgctt gcttcgggtg gcatcggaat gccggcgctg 960
caagcaatgt tgtccaggca ggtggatgag gaacgtcagg ggcagctgca aggctcactg 1020
gcggcgctca ccagcctgac ctcgatcgtc ggacccctcc tcttcacggc gatctatgcg 1080
gcttctataa caacgtggaa cgggtgggca tggattgcag gcgctgccct ctacttgctc 1140
tgcctgccgg cgctgcgtcg cgggctttgg agcggcgcag ggcaacgagc cgatcgctga 1200
Claims (5)
1. An erythromycin inducible promoter is a specific DNA molecule shown in a sequence table SEQ ID NO.1 sequence.
2. An expression cassette comprising a specific DNA molecule according to claim 1.
3. A recombinant plasmid comprising the specific DNA molecule of claim 1.
4. Use of the specific DNA molecule of claim 1, the expression cassette of claim 2, the recombinant plasmid of claim 3 for promoting expression of a gene of interest.
5. Use according to claim 4 for promoting the expression of a gene of interest, comprising the steps of: inserting a specific DNA molecule of claim 1 upstream of any gene or enhancer of interest, or inserting a gene of interest downstream of said specific DNA molecule in the expression cassette of claim 2, or inserting a gene of interest downstream of said specific DNA molecule in the recombinant plasmid of claim 3, the expression of said gene of interest being initiated by said specific DNA molecule.
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