CN116121288A - Vector for cloning pseudomonas putida large fragment DNA and application thereof - Google Patents

Vector for cloning pseudomonas putida large fragment DNA and application thereof Download PDF

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CN116121288A
CN116121288A CN202310304642.XA CN202310304642A CN116121288A CN 116121288 A CN116121288 A CN 116121288A CN 202310304642 A CN202310304642 A CN 202310304642A CN 116121288 A CN116121288 A CN 116121288A
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pbbr1
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刘惠忠
张志清
谢小保
施庆珊
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Institute of Microbiology of Guangdong Academy of Sciences
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Abstract

The invention discloses a group of vectors for cloning pseudomonas putida large fragment DNA and application thereof. The plasmid combination comprises two plasmids pBCPP51 and pBCPP52, the sequences of which are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, can effectively clone a specific DNA region in a bacterial genome, and is suitable for large fragments which are more than 10kb and difficult to amplify by a PCR method. By means of homologous recombination, two plasmids carrying homology arms are inserted into the genome at the upstream and downstream of a large fragment to be cloned in sequence, the genome is extracted, proper restriction endonuclease is selected for single enzyme digestion, then the fragment is subjected to self-interlinking to obtain plasmids containing the fragment to be cloned, and the plasmids obtained after cloning can be replicated in escherichia coli and pseudomonas putida.

Description

Vector for cloning pseudomonas putida large fragment DNA and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a group of plasmid vectors for cloning pseudomonas putida large fragment DNA and application thereof.
Background
There are several gene clusters in bacterial genome, which are closely arranged and co-transcribed, and the products of these genes can be used as different subunits to form a functional structure or to participate in different steps of transformation of a substance to jointly fulfill a biological function. In addition, there are very large individual genes, such as the adhesion protein gene labA in Pseudomonas putida, which has a sequence length of about 26kb. In studying gene function, it is often necessary to knock out the gene and then to feed back the knocked-out gene into the knocked-out strain, and observe a phenotypic change to determine the gene function; in addition, in the field of synbiotic biology, it is also necessary to clone large fragment DNA containing a plurality of key genes and introduce the fragment DNA into chassis cells for expression. For a gene of a general length, the gene can be amplified by PCR and then ligated into a plasmid, but for a DNA fragment of 10kb or more, it is often difficult for PCR to amplify a target fragment efficiently, and thus development of a novel method capable of cloning a large fragment of DNA efficiently has been desired.
Disclosure of Invention
In order to solve the technical problems, the invention provides a group of plasmids for effectively cloning large fragment DNA in pseudomonas putida genome. The plasmid combination comprises two plasmids pBCPP51 and pBCPP52, the two plasmids are respectively integrated into two ends of large fragment DNA to be cloned in pseudomonas putida genome through homologous exchange, the genome is extracted, proper restriction endonuclease is selected for single enzyme digestion, the two plasmids are cut off together with the DNA fragment to be cloned, and then the fragment is connected by using ligase to form circular DNA, wherein the circular DNA has key elements of the plasmids, such as replication origin, replication protein gene, antibiotic resistance gene and the like, and can be replicated in escherichia coli and pseudomonas putida.
The nucleotide sequence of the plasmid pBCPP51 is shown as SEQ ID NO.1, the nucleotide sequence of the plasmid pBCPP52 is shown as SEQ ID NO.2, and the physical map is shown as figure 1.
The plasmid pBCPP51 contains gentamicin resistance gene, and the plasmid pBCPP52 contains tetracycline resistance gene.
The plasmids pBCPP51 and pBCPP52 contain an R6K ori origin of replication, which is replicable in E.coli containing lambda pir but not in Pseudomonas putida.
The plasmid pBCPP51 also contains an origin of replication pBBR1 oriV from plasmid pBBR1MCS-5, after which a multiple cloning site is added, pBCPP52 contains the replication protein gene pBBR1 rep required for the origin of replication, before which a multiple cloning site is added, and when two plasmids are ligated at the multiple cloning site, the resulting circular DNA contains the origin of replication pBBR1 oriV and the corresponding replication protein gene pBBR1 rep, and thus can replicate in Pseudomonas putida.
The restriction endonuclease recognition site sequence for the multiple cloning site MCS is HindIII, pstI, salI, xbaI, bamHI, kpnI, xhoI and SacI in order.
The construction method of the plasmid comprises the following steps:
(1) The whole plasmid is reversely amplified from the junction of the replication origin pBBR1 oriV of the plasmid pBBR1MCS-5 and the replication protein gene rep to obtain a linearization plasmid, a multi-cloning site region is amplified from pUC19, an EcoRI recognition sequence is subjected to point mutation, an XhoI cleavage site is added, and the two fragments are connected through recombinase to obtain the plasmid pBBR1MCS-5MCS.
(2) Amplifying fragments containing gentamicin resistance genes, mob transfer elements, pBBR1 oriV and multiple cloning sites from pBBR1MCS-5MCS, amplifying an replication origin R6K ori from a plasmid pDS3.0, wherein the front end of the R6K ori fragment contains an EcoRI enzyme cleavage site, and connecting the two fragments through recombinase to obtain a plasmid pBCPP51, wherein host bacteria are preferably E.coli S17-1 lambda pir;
(3) A fragment containing a multiple cloning site and rep is amplified from pBBR1MCS-5MCS, a fragment containing R6Kori and RP4 junction transfer element is amplified from pDS3.0, a tetracycline resistance gene fragment is amplified from pTRG, and the three fragments are linked by a recombinase to obtain plasmid pBCPP52, the host bacterium is preferably E.coli S17-1 lambda pir.
The construction method of the plasmid comprises the following specific steps:
(1) Using pBBR1MCS-5 as a template, and using primers BBR1-fanA1 and BBR1-fanS1 to amplify to obtain a linearized plasmid; using pUC19 as a template, amplifying by using primers 19MCSs and 19MCSa to obtain multiple cloning sites, and connecting the two fragments by recombinase to obtain a plasmid pBBR1MCS-5MCS;
BBR1-fanA1：5’-ACCTGCAGGCATGCAAGCTTGCGGCACCATGCGCAATCA-3’；
BBR1-fanS1：5’-CCCGGGTACCGAGCTCGAGTGCGGCACCCTACCGCATGG-3’；
19MCSs：5’-CTCGAGCTCGGTACCCGGG-3’；
19MCSa：5’-AAGCTTGCATGCCTGCAGGT-3’；
(2) Using pBBR1MCS-5MCS as template, using primers BBR1-fanS3 and 19MCSs to amplify to obtain fragment containing gentamicin resistance gene, mob transfer element, pBBR1 oriV and multiple cloning site; amplifying by using pDS3.0 as a template and using the primers R6Ks1 and R6Ka1 to obtain a fragment of the replication origin R6K ori; connecting the two fragments through recombinase to obtain plasmid pBCPP51;
BBR1-fanS3：5’-AACGGCTGACATGGGAATTCGGACGCACACCGTGGAAAC-3’；
19MCSs：5’-CTCGAGCTCGGTACCCGGG-3’；
R6Ks1：5’-GAATTCCCATGTCAGCCGTT-3’；
R6Ka1：5’-CCCGGGTACCGAGCTCGAGACGATGCGTCCGGCGTAGAG-3’；
(3) Using pBBR1MCS-5MCS as template, using primers BBR1-fanA2 and 19MCSa to amplify fragment containing multiple cloning site and rep; amplifying by using pDS3.0 as a template and using the primers R6Ks2 and RP4a2 to obtain a fragment containing the replication origin R6Kori and the RP4 junction transfer element; the pTRG is used as a template, primers tetS and tetA are used for amplification to obtain tetracycline resistance gene fragments, and the three fragments are connected through recombinase to obtain plasmid pBCPP52.
BBR1-fanA2：5’-CGTGCTCCTGTCGTTGAGGATTGCTCAGGCTCTCCCCGT-3’；
19MCSa：5’-AAGCTTGCATGCCTGCAGGT-3’；
R6Ks2：5’-GTCAAAAATAAGTGCCTTCCGAATTCCCATGTCAGCCGTT-3’；
RP4a2：5’-ACCTGCAGGCATGCAAGCTTTCTGCTCTGATGCCGCATAG-3’；
tetS：5’-GGAAGGCACTTATTTTTGAC-3’；
tetA：5’-TCCTCAACGACAGGAGCACG-3’。
The plasmid combination of the invention can be applied to cloning large fragment DNA in pseudomonas putida genome.
A method for cloning large fragment DNA in pseudomonas putida genome using said plasmid combination comprising the steps of:
(1) The front and rear ends of the DNA region to be cloned were amplified as homology arms for the upstream and downstream homology exchange, and respectively joined into pBCPP51 and pBCPP52 digested with EcoRI, and E.coli S17-1. Lambda. Pir was transformed to obtain plasmids pBCPP51up and pBCPP52dn, respectively.
(2) pBCPP51up was introduced into Pseudomonas putida by conjugal transfer, and integrated into the genome at the position of the upstream homology arm by homology exchange, at which time Pseudomonas putida acquired gentamicin resistance, and the primary integrated strain was obtained by screening on a resistance plate.
(3) pBCPP52dn was introduced into the primary integrated strain by conjugal transfer, and integrated into the genome by homology exchange at the position of the downstream homology arm, at which time the primary integrated strain acquired tetracycline resistance, and the secondary integrated strain was obtained by screening on the resistance plate.
(4) Extracting genome of the secondary integrated strain, and selecting one enzyme cutting site in multiple cloning sites MCS on pBCPP51 and pBCPP52 for enzyme cutting, wherein the enzyme cutting site cannot exist on large-fragment DNA to be cloned, recovering large-fragment DNA enzyme cutting products through agarose gel electrophoresis and gel cutting, and removing small-fragment enzyme cutting products is beneficial to the smooth proceeding of subsequent experiments.
(5) The large fragment DNA recovered by the excision of the well is ligated using T4 ligase, preferably by incubating for 12h at 16℃and then incubating for 12h at 4 ℃. Transforming the ligation product into E.coli competent cells, preferably E.coli XL10-Gold heat shock competent cells suitable for transformation of large plasmids and precious ligation products, coating a resistance plate containing gentamicin and tetracycline, picking single colonies for verification, and obtaining plasmids sequentially comprising the elements pBBR1 oriV, mob, gentamicin resistance gene, large fragment DNA to be cloned, tetracycline resistance gene and pBBR1 rep on which pBBR1 oriV replication depends (FIG. 2); the plasmid can also replicate in Pseudomonas putida, and can be introduced into Pseudomonas putida by electrotransformation.
Drawings
FIG. 1 is a map of plasmids pBCPP51 and pBCPP52.
FIG. 2 is a schematic diagram showing the steps and principles of cloning large genomic DNA using plasmids pBCPP51 and pBCPP52.
FIG. 3 shows transformants obtained by transforming E.coli XL10-Gold with pBCPP-lapA. The XbaI digested double-inserted Pseudomonas putida KT2440 genome DNA is connected by T4 ligase to transform E.coli XL10-Gold competent cells, and LB solid plates containing 20 mug/mL gentamicin and 20 mug/mL tetracycline are coated.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1: construction of plasmids pBCPP51 and pBCPP52
(1) Primers BBR1-fanA1 and BBR1-fanS1 were designed and a linearized plasmid of about 4.8kb was amplified using Fidelity enzymes using pBBR1MCS-5 (GenBank: U25061.1) as template according to the following procedure: 95 ℃ for 1min;95 ℃ for 20s,55 ℃ for 20s,72 ℃ for 3min,30 cycles; and at 72℃for 5min. Primers 19MCSs and 19MCSa were designed and 54bp of multiple cloning sites were amplified using a fidelity enzyme using pUC19 (GenBank: M77789.2) as template according to the following procedure: 95 ℃ for 1min;95℃for 20s,56℃for 20s,34 cycles. And (3) carrying out agarose electrophoresis and gel cutting recovery on the two amplified products, then connecting fragments by using a recombinant cloning kit (nuuzan), converting competent cells of escherichia coli S17-1 lambda pir by heat shock, coating an LB solid plate containing 20 mug/mL, culturing at 37 ℃, picking single colony and then carrying out sequencing identification on the positions of multiple cloning sites, and finally obtaining pBBR1MCS-5MCS.
The primer sequences are as follows:
BBR1-fanA1：5’-ACCTGCAGGCATGCAAGCTTGCGGCACCATGCGCAATCA-3’；
BBR1-fanS1：5’-CCCGGGTACCGAGCTCGAGTGCGGCACCCTACCGCATGG-3’；
19MCSs：5’-CTCGAGCTCGGTACCCGGG-3’；
19MCSa：5’-AAGCTTGCATGCCTGCAGGT-3’。
(2) Using primers BBR1-fanS3 and 19MCSs and pBBR1MCS-5MCS as templates, a fragment of about 2.9kb was amplified using a Fidelity enzyme according to the following procedure: 95 ℃ for 1min;95℃20s,55℃20s,72℃2min,30 cycles; and at 72℃for 5min. Using primers R6Ks1 and R6Ka1 and pDS3.0 (nucleotide sequence shown in SEQ ID NO. 3) as templates, a fragment of about 0.4kb was amplified using a Fidelity enzyme according to the following procedure: 95 ℃ for 1min;95℃20s,55℃20s,72℃20s,34 cycles; and at 72℃for 5min. And (3) carrying out agarose electrophoresis and gel cutting recovery on the two amplified products, then connecting fragments by using a recombinant cloning kit (Norfluzab), carrying out heat shock transformation on competent cells of escherichia coli S17-1 lambda pir, coating an LB solid plate containing 20 mug/mL gentamicin, culturing at 37 ℃, picking single colony, carrying out PCR detection, and carrying out sequencing identification on correct clone to finally obtain pBCPP51 (figure 1), wherein the sequence is shown as SEQ ID NO. 1.
The primer sequences are as follows:
BBR1-fanS3：5’-AACGGCTGACATGGGAATTCGGACGCACACCGTGGAAAC-3’；
19MCSs：5’-CTCGAGCTCGGTACCCGGG-3’；
R6Ks1：5’-GAATTCCCATGTCAGCCGTT-3’；
R6Ka1：5’-CCCGGGTACCGAGCTCGAGACGATGCGTCCGGCGTAGAG-3’。
the sequence of pDS3.0 is shown as SEQ ID NO.3, and specifically comprises the following steps:
TTGTTATCCGCTCACAATTCCACATGTGGAATTCCACATGTGGAATTCCCATGTCAGCCGTTAAGTGTTCCTGTGTCACTCAAAATTGCTTTGAGAGGCTCTAAGGGCTTCTCAGTGCGTTACATCCCTGGCTTGTTGTCCACAACCGTTAAACCTTAAAAGCTTTAAAAGCCTTATATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTTAGCCATGAGAGCTTAGTACGTTAGCCATGAGGGTTTAGTTCGTTAAACATGAGAGCTTAGTACGTTAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTACTATCAACAGGTTGAACTGCTGATCTTCAGATCCTCTACGCCGGACGCATCGTGGCCGGATCCAGCCGACCAGGCTTTCCACGCCCGCGTGCCGCTCCATGTCGTTCGCGCGGTTCTCGGAAACGCGCTGCCGCGTTTCGTGATTGTCACGCTCAAGCCCGTAGTCCCGTTCGAGCGTCGCGCAGAGGTCAGCGAGGGCGCGGTAGGCCCGATACGGCTCATGGATGGTGTTTCGGGTCGGGTGAATCTTGTTGATGGCGATATGGATGTGCAGGTTGTCGGTGTCGTGATGCACGGCACTGACGCGCTGATGCTCGGCGAAGCCAAGCCCAGCGCAGATGCGGTCCTCAATCGCGCGCAACGTCTCCGCGTCGGGCTTCTCTCCCGCGCGGAAGCTAACCAGCAGGTGATAGGTCTTGTCGGCCTCGGAACGGGTGTTGCCGTGCTGGGTCGCCATCACCTCGGCCATGACAGCGGGCAGGGTGTTTGCCTCGCAGTTCGTGACGCGCACGTGACCCAGGCGCTCGGTCTTGCCTTGCTCGTCGGTGATGTACTTCACCAGCTCCGCGAAGTCGCTCTTCTTGATGGAGCGCATGGGGACGTGCTTGGCAATCACGCGCACCCCCCGGCCGTTTTAGCGGCTAAAAAAGTCATGGCTCTGCCCTCGGGCGGACCACGCCCATCATGACCTTGCCAAGCTCGTCCTGCTTCTCTTCGATCTTCGCCAGCAGGGCGAGGATCGTGGCATCACCGAACCGCGCCGTGCGCGGGTCGTCGGTGAGCCAGAGTTTCAGCAGGCCGCCCAGGCGGCCCAGGTCGCCATTGATGCGGGCCAGCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGG
GCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTAC
GCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGG
TGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCAC
CTATCCTGCCCGGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTT
TGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTA
TAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTG
TGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGG
ACAGGCGAGAGACGATGCCAAAGAGCTACACCGACGAGCTGGCCGAGTGGGTTG
AATCCCGCGCGGCCAAGAAGCGCCGGCGTGATGAGGCTGCGGTTGCGTTCCTGGC
GGTGAGGGCGGATGTCGAGGCGGCGTTAGCGTCCGGCTATGCGCTCGTCACCATTT
GGGAGCACATGCGGGAAACGGGGAAGGTCAAGTTCTCCTACGAGACGTTCCGCTC
GCACGCCAGGCGGCACATCAAGGCCAAGCCCGCCGATGTGCCCGCACCGCAGGCC
AAGGCTGCGGAACCCGCGCCGGCACCCAAGACGCCGGAGCCACGGCGGCCGAAG
CAGGGGGGCAAGGCTGAAAAGCCGGCCCCCGCTGCGGCCCCGACCGGCTTCACCT
TCAACCCAACACCGGACAAAAAGGATCCTCTACGCCGGACGCATCGTGGCCGGCA
TCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGG
GAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGT
GGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCTGCCTCGCGCGT
TTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAG
CTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGG
TGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTG
TATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAA
TCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG
TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCC
CGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA
TGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCC
AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG
TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCG
CAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGG
CTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTG
TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC
CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCC
ATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT
AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGC
GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA
AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC
ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAA
CAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA
TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA
TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG
CACATTTCCCCGAAAAGTGCCACCTGCAGTTCACTTACACCGCTTCTCAACCCGGT
ACGCACCAGAAAATCATTGATATGGCCATGAATGGCGTTGGATGCCGGGCAACAG
CCCGCATTATGGGCGTTGGCCTCAACACGATTTTACGTCACTTAAAAAACTCAGGC
CGCAGTCGGTAACCTCGCGCATACAGCCGGGCAGTGACGTCATCGTCTGCGCGGA
AATGGACGAACAGTGGGGCTATGTCGGGGCTAAATCGCGCCAGCGCTGGCTGTTT
TACGCGTATGACAGTCTCCGGAAGACGGTTGTTGCGCACGTATTCGGTGAACGCAC
TATGGCGACGCTGGGGCGTCTTATGAGCCTGCTGTCACCCTTTGACGTGGTGATAT
GGATGACGGATGGCTGGCCGCTGTATGAATCCCGCCTGAAGGGAAAGCTGCACGT
AATCAGCAAGCGATATACGCAGCGAATTGAGCGGCATAACCTGAATCTGAGGCAG
CACCTGGCACGGCTGGGACGGAAGTCGCTGTCGTTCTCAAAATCGGTGGAGCTGC
ATGACAAAGTCATCGGGCATTATCTGAACATAAAACACTATCAATAAGTTGGAGT
CATTACCAAAAGGTTAGGAATACGGTTAGCCATTTGCCTGCTTTTATATAGTTCAT
ATGGGATTCACCTTTATGTTGATAAGAAATAAAAGAAAATGCCAATAGGATATCG
GCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTGTTCAAGGATGCTG
TCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTTGGCGCAAA
CGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGAC
ATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGT
TAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGG
CCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGC
CGTCAATCGTCATTTTTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTC
ATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCAATCAGC
GGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCG
CCGTTTGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCT
TGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTTGTTAAATAAAGATTCTTC
GCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTGTGGCC
TTTATCTTCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCTGTAGTT
GCCTTCATCGATGAACTGCTGTACATTTTGATACGTTTTTCCGTCACCGTCAAAGAT
TGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGTCTGATGCTGATACGTT
AACTTGTGCAGTTGTCAGTGTTTGTTTGCCGTAATGTTTACCGGAGAAATCAGTGT
AGAATAAACGGATTTTTCCGTCAGATGTAAATGTGGCTGAACCTGACCATTCTTGT
GTTTGGTCTTTTAGGATAGAATCATTTGCATCGAATTTGTCGCTGTCTTTAAAGACG
CGGCCAGCGTTTTTCCAGCTGTCAATAGAAGTTTCGCCGACTTTTTGATAGAACAT
GTAAATCGATGTGTCATCCGCATTTTTAGGATCTCCGGCTAATGCAAAGACGATGT
GGTAGCCGTGATAGTTTGCGACAGTGCCGTCAGCGTTTTGTAATGGCCAGCTGTCC
CAAACGTCCAGGCCTTTTGCAGAAGAGATATTTTTAATTGTGGACGAATCGAATTC
AGGAACTTGATATTTTTCATTTTTTTGCTGTTCAGGGATTTGCAGCATATCATGGCG
TGTAATATGGGAAATGCCGTATGTTTCCTTATATGGCTTTTGGTTCGTTTCTTTCGC
AAACGCTTGAGTTGCGCCTCCTGCCAGCAGTGCGGTAGTAAAGGTTAATACTGTTG
CTTGTTTTGCAAACTTTTTGATGTTCATCGTTCATGTCTCCTTTTTTATGTACTGTGT
TAGCGGTCTGCTTCTTCCAGCCCTCCTGTTTGAAGATGGCAAGTTAGTTACGCACA
ATAAAAAAAGACCTAAAATATGTAAGGGGTGACGCCAAAGTATACACTTTGCCCT
TTACACATTTTAGGTCTTGCCTGCTTTATCAGTAACAAACCCGCGCGATTTACTTTT
CGACCTCATTCTATTAGACTCTCGTTTGGATTGCAACTGGTCTATTTTCCTCTTTTGT
TTGATAGAAAATCATAAAAGGATTTGCAGACTACGGGCCTAAAGAACTAAAAAAT
CTATCTGTTTCTTTTCATTCTCTGTATTTTTTATAGTTTCTGTTGCATGGGCATAAAG
TTGCCTTTTTAATCACAATTCAGAAAATATCATAATATCTCATTTCACTAAATAATA
GTGAACGGCAGGTATATGTGATGGGTTAAAAAGGATCGATCCTCTAGAGTCGACG
CGTCGATCCGGTGATTGATTGAGCAAGCTAGCTTTATGCTTGTAAACCGTTTTGTG
AAAAAATTTTTAAAATAAAAAAGGGGACCTCTAGGGTCCCCAATTAATTAGTAAT
ATAATCTATTAAAGGTCATTCAAAAGGTCATCCACCGGATCAATTCCCCTGCTCGC
GCAGGCTGGGTGCCAAGCTCTCGGGTAACATCAAGGCCCGATCCTTGGAGCCCTTG
CCCTCCCGCACGATGATCGTGCCGTGATCGAAATCCAGATCCTTGACCCGCAGTTG
CAAACCCTCACTGATCCGCGCGTCGAATTGCCGGGAAGCCGATCTCGGCTTGAACG
AATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTAT
GCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGT
AGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGC
GGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAG
ATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAA
CAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAA
GTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGA
ACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCC
GAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGC
GACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAA
CATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATA
GACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTAC
CACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTG
CATTACAGTTTACGAACCGAACAGGCTTATGTCAATTCGAGAATTAATTCGACGCG
CAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGT
CGGCAAATAATGTCTAACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTA
ACTCAAGCGTTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCA
AGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGAT
ATATCATGAAAGGCTGGCTTTTTCTTGTTATCGCAATAGTTGGCGAAGTAATCGCA
ACATCCGCATTAAAATCTAGCGAGGGCTTTACTAAGCTGATCCGGTGGATGACCTT
TTGAATGACCTTTAATAGATTATATTACTAATTAATTGGGGACCCTAGAGGTCCCC
TTTTTTATTTTAAAAATTTTTTCACAAAACGGTTTACAAGCATAAAGCTAGCTTGCT
CAATCAATCACCGGATCGACGCGTCGACGGATCCCAAGCTTCTTCTAGAGGTACCGCATGCGATATCGAGCTCTCCCGGGAATTCCACAAA。
the sequence of pBCPP51 is shown in SEQ ID NO.1, specifically:
CTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCTCCTGCGG
CGGCCTGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCGGCCACG
GCTTCCGGCGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGCGGTGCA
TAGGCGCGTGGCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGCCGCTC
CAGGGCCTCGTAGAACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATGCCCC
GTTGCAGCCCTAGATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATCTGC
GCTTTGTTGCCGATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACCAC
GAACGCGGTCATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGAT
CCGCCCCGTACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGT
TCTTGGCTGGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAA
CACAGCGTCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCAT
CGGTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGC
GTCTCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAG
CTTCTTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACC
GGCTCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTAT
ACTCACTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACG
GGCTTGCTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGATA
TGTGGACGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGACAA
CCCTGCTGGACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGGGAT
TGCCCACCGGCTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGCCTGC
GGCCTTGCCCCATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCGGCCTG
CGCGCTTCGCTTGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCGCAGCGA
CCGCGCAGCGGCTTGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGAGTGGGGG
CAGTCGAAGGCGAAGCCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCGAGTGCGGGG
GTTCCAAGGGGGCAGCGCCACCTTGGGCAAGGCCGAAGGCCGCGCAGTCGATCAACAA
GCCCCGGAGGGGCCACTTTTTGCCGGAGGGGGAGCCGCGCCGAAGGCGTGGGGGAAC
CCCGCAGGGGTGCCCTTCTTTGGGCACCAAAGAACTAGATATAGGGCGAAATGCGAAAG
ACTTAAAAATCAACAACTTAAAAAAGGGGGGTACGCAACAGCTCATTGCGGCACCCCC
CGCAATAGCTCATTGCGTAGGTTAAAGAAAATCTGTAATTGACTGCCACTTTTACGCAAC
GCATAATTGTTGTCGCGCTGCCGAAAAGTTGCAGCTGATTGCGCATGGTGCCGCAAGCT
TGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAGACGATGCGTC
CGGCGTAGAGGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTACGTACTAAGCTCTCA
TGTTTCACGTACTAAGCTCTCATGTTTAACGTACTAAGCTCTCATGTTTAACGAACTAAA
CCCTCATGGCTAACGTACTAAGCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCACGT
ACTAAGCTCTCATGTTTGAACAATAAAATTAATATAAATCAGCAACTTAAATAGCCTCTAA
GGTTTTAAGTTTTATAAGAAAAAAAAGAATATATAAGGCTTTTAAAGCTTTTAAGGTTTA
ACGGTTGTGGACAACAAGCCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCCTCTCA
AAGCAATTTTGAGTGACACAGGAACACTTAACGGCTGACATGGGAATTCGGACGCACA
CCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGTTCGGTTCGTAAACT
GTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCG
GTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTAT
GCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGA
GCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAA
CAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAA
GTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACC
TACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTC
ATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCT
GCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGA
GCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACG
CGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTC
TCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACC
GCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGCGGAGTTGTTCGGTAA
ATTGTCACAACGCCGCCAGGTGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTTCCTGC
TGGCGCTGGGCCTGTTTCTGGCGCTGGACTTCCCGCTGTTCCGTCAGCAGCTTTTCGCC
CACGGCCTTGATGATCGCGGCGGCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCTTCAGGCGCTCCCGAAGGT。
(3) Using primers BBR1-fanA2 and 19MCSa and pBBR1MCS-5MCS as templates, an approximately 1kb fragment was amplified using a fidelity enzyme according to the following procedure: 95 ℃ for 1min;95℃20s,55℃20s,72℃1min,34 cycles; and at 72℃for 5min. Using primers R6Ks2 and RP4a2 as template pDS3.0, an approximately 2.5kb fragment was amplified using fidelity enzyme following the following procedure: 95 ℃ for 1min;95℃20s,55℃20s,72℃2min,30 cycles; and at 72℃for 5min. Using the primers tetS and tetA as templates for pTRG (GenBank: AF 361441.1), a tetracycline resistance fragment of about 1.5kb was amplified using a fidelity enzyme according to the following procedure: 95 ℃ for 1min;95℃20s,55℃20s,72℃1min,34 cycles; and at 72℃for 5min. And (3) carrying out agarose electrophoresis and gel cutting recovery on the three amplified products, then connecting fragments by using a recombinant cloning kit (nuuzan), carrying out heat shock transformation on competent cells of escherichia coli S17-1 lambda pir, coating an LB solid plate containing 20 mug/mL tetracycline, culturing at 37 ℃, picking single colony, carrying out PCR detection, and carrying out sequencing identification on correct clone to finally obtain pBCPP52 (figure 1), wherein the sequence is shown as SEQ ID NO. 2.
The primer sequences are as follows:
BBR1-fanA2：5’-CGTGCTCCTGTCGTTGAGGATTGCTCAGGCTCTCCCCGT-3’；
19MCSa：5’-AAGCTTGCATGCCTGCAGGT-3’；
R6Ks2：5’-GTCAAAAATAAGTGCCTTCCGAATTCCCATGTCAGCCGTT-3’；
RP4a2：5’-ACCTGCAGGCATGCAAGCTTTCTGCTCTGATGCCGCATAG-3’；
tetS：5’-GGAAGGCACTTATTTTTGAC-3’；
tetA：5’-TCCTCAACGACAGGAGCACG-3’。
the sequence of pBCPP52 is shown in SEQ ID NO.2, specifically:
AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAGTGCGGCACCCTACCGCATGGAGATAAGCATGGCCACGCAGTCCAGAGAAATCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGGTGCAAACGGAACGCAAAGCGCATGAGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGGCGGCAATGCTGCTGCATCACCTCGTGGCGCAGATGGGCCACCAGAACGCCGTGGTGGTCAGCCAGAAGACACTTTCCAAGCTCATCGGACGTTCTTTGCGGACGGTCCAATACGCAGTCAAGGACTTGGTGGCCGAGCGCTGGATCTCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGTCGGCCTACGTGGTCAATGACCGCGTG
GCGTGGGGCCAGCCCCGCGACCAGTTGCGCCTGTCGGTGTTCAGTGCCGCCGTGGTGG
TTGATCACGACGACCAGGACGAATCGCTGTTGGGGCATGGCGACCTGCGCCGCATCCCG
ACCCTGTATCCGGGCGAGCAGCAACTACCGACCGGCCCCGGCGAGGAGCCGCCCAGCC
AGCCCGGCATTCCGGGCATGGAACCAGACCTGCCAGCCTTGACCGAAACGGAGGAATG
GGAACGGCGCGGGCAGCAGCGCCTGCCGATGCCCGATGAGCCGTGTTTTCTGGACGAT
GGCGAGCCGTTGGAGCCGCCGACACGGGTCACGCTGCCGCGCCGGTAGCACTTGGGTT
GCGCAGCAACCCGTAAGTGCGCTGTTCCAGACTATCGGCTGTAGCCGCCTCGCCGCCCT
ATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCT
GAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCGTTTTTATCAGGCTCTGGGAGG
CAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAATCCTCA
ACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCG
CCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTT
GCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGT
TAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCTCGAC
GCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTT
CCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAG
TTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCT
GCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAAT
CATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGC
GCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGG
ACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG
CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTG
CCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATA
GTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATGG
GTCGGCGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGG
CCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGTAAGGAGATGGCGCCCAACAGTC
CCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAA
GTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCAC
CTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGAATCCACAGGACGGG
TGTGGTCGCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTG
GGCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCAT
CAACGCATATAGCGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGA
TATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGG
GTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACT
GCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAATCGATGATAAGCTGTCA
AACATGAGAATTGAGCGAACGTCAAAAATAAGTGCCTTCCGAATTCCCATGTCAGCCGT
TAAGTGTTCCTGTGTCACTCAAAATTGCTTTGAGAGGCTCTAAGGGCTTCTCAGTGCGTT
ACATCCCTGGCTTGTTGTCCACAACCGTTAAACCTTAAAAGCTTTAAAAGCCTTATATATT
CTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATT
TTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTTAGCCATG
AGAGCTTAGTACGTTAGCCATGAGGGTTTAGTTCGTTAAACATGAGAGCTTAGTACGTTA
AACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACGTACTATCAACAGGTTGAA
CTGCTGATCTTCAGATCCTCTACGCCGGACGCATCGTGGCCGGATCCAGCCGACCAGGC
TTTCCACGCCCGCGTGCCGCTCCATGTCGTTCGCGCGGTTCTCGGAAACGCGCTGCCGC
GTTTCGTGATTGTCACGCTCAAGCCCGTAGTCCCGTTCGAGCGTCGCGCAGAGGTCAGC
GAGGGCGCGGTAGGCCCGATACGGCTCATGGATGGTGTTTCGGGTCGGGTGAATCTTGT
TGATGGCGATATGGATGTGCAGGTTGTCGGTGTCGTGATGCACGGCACTGACGCGCTGA
TGCTCGGCGAAGCCAAGCCCAGCGCAGATGCGGTCCTCAATCGCGCGCAACGTCTCCG
CGTCGGGCTTCTCTCCCGCGCGGAAGCTAACCAGCAGGTGATAGGTCTTGTCGGCCTCG
GAACGGGTGTTGCCGTGCTGGGTCGCCATCACCTCGGCCATGACAGCGGGCAGGGTGT
TTGCCTCGCAGTTCGTGACGCGCACGTGACCCAGGCGCTCGGTCTTGCCTTGCTCGTCG
GTGATGTACTTCACCAGCTCCGCGAAGTCGCTCTTCTTGATGGAGCGCATGGGGACGTG
CTTGGCAATCACGCGCACCCCCCGGCCGTTTTAGCGGCTAAAAAAGTCATGGCTCTGCC
CTCGGGCGGACCACGCCCATCATGACCTTGCCAAGCTCGTCCTGCTTCTCTTCGATCTTC
GCCAGCAGGGCGAGGATCGTGGCATCACCGAACCGCGCCGTGCGCGGGTCGTCGGTGA
GCCAGAGTTTCAGCAGGCCGCCCAGGCGGCCCAGGTCGCCATTGATGCGGGCCAGCTC
GCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCA
GGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGT
CTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCA
GCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGG
ATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTC
GCGGGTGGGCCTACTTCACCTATCCTGCCCGGCTGACGCCGTTGGATACACCAAGGAAA
GTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGA
AAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTG
CTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGA
GGGGACAGGCGAGAGACGATGCCAAAGAGCTACACCGACGAGCTGGCCGAGTGGGTT
GAATCCCGCGCGGCCAAGAAGCGCCGGCGTGATGAGGCTGCGGTTGCGTTCCTGGCGG
TGAGGGCGGATGTCGAGGCGGCGTTAGCGTCCGGCTATGCGCTCGTCACCATTTGGGAG
CACATGCGGGAAACGGGGAAGGTCAAGTTCTCCTACGAGACGTTCCGCTCGCACGCCA
GGCGGCACATCAAGGCCAAGCCCGCCGATGTGCCCGCACCGCAGGCCAAGGCTGCGGA
ACCCGCGCCGGCACCCAAGACGCCGGAGCCACGGCGGCCGAAGCAGGGGGGCAAGGC
TGAAAAGCCGGCCCCCGCTGCGGCCCCGACCGGCTTCACCTTCAACCCAACACCGGAC
AAAAAGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCG
GTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGG
GCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGT
TGGGCGCCATCTCCTTGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACA
CATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAA
GCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGA。
example 2: the procedure and schematic diagram of cloning large fragment DNA of genome by using plasmids pBCPP51 and pBCPP52 in cloning DNA region of adhesion protein gene lapA in Pseudomonas putida KT2440 genome are shown in figure 2.
(1) The genomic sequence of Pseudomonas putida KT2440 was obtained from NCBI database (NC_ 002947.4), the size of the lapA gene was 26kb, and to increase the length of the cloned fragment in this example, the fragment was extended back and forth to about 53kb in total, and upstream and downstream homology arm fragments of about 0.8kb were amplified using primers pairs lapUPS/lapUPA and lapADnS/lapADnA, respectively, using KT2440 genomic DNA as templates, using Fidelity enzymes according to the following procedure: 95 ℃ for 1min;95℃20s,55℃20s,72℃1min,34 cycles; and at 72℃for 5min. The preparation method comprises the steps of carrying out single enzyme digestion on pBCPP51 and pBCPP52 by using EcoRI, carrying out gel digestion recovery on the PCR products and enzyme digestion products, connecting an upstream homology arm into the pBCPP51 by using a recombinant cloning kit (Norfirazan), connecting a downstream homology arm into the pBCPP52, carrying out heat shock transformation on competent cells of escherichia coli S17-1 lambda pir, respectively coating LB solid plates containing 20 mug/mL gentamicin and 20 mug/mL tetracycline, culturing at 37 ℃, picking single colonies, and carrying out PCR verification to obtain pBCPP 51-lapeup and pBCPP52-lapAdn.
The primer sequences are as follows:
lapAupS：5’-TCCACGGTGTGCGTCCGAATTCCAAGGAAAAGCGCCACTGGT-3’；
lapAupA：5’-TTAACGGCTGACATGGGAATTCCCCTGACCACCAAGAAGCCA-3’；
lapAdnS：5’-TTAACGGCTGACATGGGAATTCCTTGGGGGATTAGCGAGACG-3’；
lapAdnA：5’-AAAATAAGTGCCTTCCGAATTCCAAGCTGCTGGGCTTTGAAG-3’。
(2) After activating Escherichia coli S17-1 lambda pir containing pBCPP 51-lapeup and Pseudomonas putida KT2440 (recipient bacteria), respectively centrifugally collecting thalli, suspending the thalli in LB liquid medium without antibiotics, mixing donor bacteria and recipient bacteria according to a volume ratio of 3:1, incubating for 12 hours in a 28 ℃ incubator, taking 100 mu L of the mixture, coating the mixture on an LB solid plate containing 25 mu g/mL chloramphenicol and 40 mu g/mL gentamicin, placing the mixture at 28 ℃ for culturing, taking single colony, and using primers lapeupyzS and lapeupyzA for PCR verification, wherein colonies without amplification products are pBCPP 51-lapeup to be integrated into an upstream target area, and are correct primary integrated strains.
The primer sequences are as follows:
lapAupyzS：5’-GGTAGAGCACTCCAGCCCG-3’；
lapAupyzA：5’-CGGCACCTACAGCACCATG-3’。
(3) After activated cultivation of E.coli S17-1 lambda pir containing pBCPP52-lapAdn and once integrated Pseudomonas putida KT2440 strain (recipient strain), the cells were collected by centrifugation, respectively, suspended in LB liquid medium without antibiotics, donor and recipient bacteria were mixed at a volume ratio of 3:1, incubated in a 28℃incubator for 12h, 100. Mu.L of the mixture was plated on LB solid plates containing 25. Mu.g/mL chloramphenicol, 40. Mu.g/mL gentamicin and 20. Mu.g/mL tetracycline, placed in 28℃for cultivation, and after single colony selection, PCR was performed using primers lapadnyzS and lapadnyzA, and colony without amplified product was pBCPP52-lapAdn integrated into the downstream target area, which was the correct secondary integrated strain.
The primer sequences are as follows:
lapAdnyzS：5’-GGTCTGCACCGTTTTTGAAA-3’；
lapAdnyzA：5’-CTATGAGCGCCTGGTCGACT-3’。
(4) Culturing a secondarily integrated pseudomonas putida KT2440 strain, collecting 5mL of bacterial liquid, extracting genome DNA by using a bacterial genome DNA extraction kit (a biological organism), and performing enzyme digestion on the genome by using the following system: 50. Mu.L of genomic DNA, 4. Mu.LXbaI restriction endonuclease (Takara), 6. Mu.L of digestion buffer, and incubation at 37℃for 6h. And (3) agarose gel electrophoresis is carried out on the enzyme digestion products, and large-fragment DNA with a wider range at the upper end of the gel block is cut out, purified and recovered to remove smaller enzyme digestion DNA fragments. The recovered cleavage products were subjected to enzymatic ligation using the following system: 20. Mu.L of digested genomic DNA (about 2. Mu.g), 4. Mu. L T4 ligase (Takara), 4. Mu.L of 10 Xligase buffer, 12. Mu.L of ddH 2 O, after 12h incubation at 16 ℃, the mixture is transferred to 4 ℃ for 12h incubation. 4 tubes of efficient E.coli XL10-Gold heat shock competent cells (organisms only) were taken, 10 mu L of the ligation product was added to each tube, mixed and incubated on ice for 30min, heat shock was performed on a 42 ℃ water bath for 35s, quickly placed back on ice and left for 2min, after 1.5h of resuscitative culture with 700 mu L of fresh LB medium, coated on LB solid plates containing 20 mu g/mL gentamicin and 20 mu g/mL tetracycline (FIG. 3), placed on 37 ℃ for culture, single colony culture was selected and PCR verification was performed, primers were lapayzS and lapayzA, the products were about 2kb, and plasmid pBCPP-lapA was obtained. The plasmid pBCPP-lapA can replicate in E.coli and Pseudomonas putida.
The primer sequences are as follows:
lapAyzS：5'-CGTTCAACATCGCTACCCTC-3'；
lapAyzA：5'-GCTCTTGCCATTCTTCCACA-3'。
the foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (10)

1. A plasmid combination comprising plasmid pBCPP51 having the sequence shown in SEQ ID No.1 and plasmid pBCPP52 having the sequence shown in SEQ ID No. 2.
2. The plasmid combination of claim 1, wherein plasmid pBCPP51 contains an R6K ori origin of replication, a gentamicin resistance gene, a conjugative transfer element mob, and an origin of replication pBBR1 oriV from pBBR1MCS-5, followed by a multiple cloning site MCS; plasmid pBCPP52 contains the tetracycline resistance gene, R6K ori origin of replication, junction transfer element RP4, pBBR1 rep, the replication protein gene pBBR1 rep required for pBBR1 oriV replication, the prep. of pBBR1 rep being ligated with the multiple cloning site MCS.
3. The plasmid combination of claim 2, wherein the multiple cloning site MCS comprises a restriction endonuclease recognition site sequence of HindIII, pstI, salI, xbaI, bamHI, kpnI, xhoI and SacI in sequence.
4. A recombinant bacterium comprising the plasmid combination of claim 1.
5. The recombinant bacterium according to claim 4, wherein the host bacterium of the recombinant bacterium is E.coli S17-1. Lambda. Pir.
6. A method of constructing a plasmid combination according to claim 1, comprising the steps of:
(1) Using pBBR1MCS-5 as a template, and using primers BBR1-fanA1 and BBR1-fanS1 to amplify to obtain a linearized plasmid; using pUC19 as a template, amplifying by using primers 19MCSs and 19MCSa to obtain multiple cloning sites, and connecting the two fragments by recombinase to obtain a plasmid pBBR1MCS-5MCS;
(2) Using pBBR1MCS-5MCS as template, using primers BBR1-fanS3 and 19MCSs to amplify to obtain fragment containing gentamicin resistance gene, mob transfer element, pBBR1 oriV and multiple cloning site; amplifying by using pDS3.0 as a template and using the primers R6Ks1 and R6Ka1 to obtain a fragment of the replication origin R6K ori; connecting the two fragments through recombinase to obtain plasmid pBCPP51;
(3) Using pBBR1MCS-5MCS as template, using primers BBR1-fanA2 and 19MCSa to amplify fragment containing multiple cloning site and rep; amplifying by using pDS3.0 as a template and using the primers R6Ks2 and RP4a2 to obtain a fragment containing the replication origin R6Kori and the RP4 junction transfer element; the pTRG is used as a template, primers tetS and tetA are used for amplification to obtain tetracycline resistance gene fragments, and the three fragments are connected through recombinase to obtain plasmid pBCPP52.
7. The construction method according to claim 6, wherein,
the primer in the step (1) is specifically:
BBR1-fanA1：5’-ACCTGCAGGCATGCAAGCTTGCGGCACCATGCGCAATCA-3’；
BBR1-fanS1：5’-CCCGGGTACCGAGCTCGAGTGCGGCACCCTACCGCATGG-3’；
19MCSs：5’-CTCGAGCTCGGTACCCGGG-3’；
19MCSa：5’-AAGCTTGCATGCCTGCAGGT-3’；
the primer in the step (2) is specifically:
BBR1-fanS3：5’-AACGGCTGACATGGGAATTCGGACGCACACCGTGGAAAC-3’；
19MCSs：5’-CTCGAGCTCGGTACCCGGG-3’；
R6Ks1：5’-GAATTCCCATGTCAGCCGTT-3’；
R6Ka1：5’-CCCGGGTACCGAGCTCGAGACGATGCGTCCGGCGTAGAG-3’
the primer in the step (3) is specifically:
BBR1-fanA2：5’-CGTGCTCCTGTCGTTGAGGATTGCTCAGGCTCTCCCCGT-3’；
19MCSa：5’-AAGCTTGCATGCCTGCAGGT-3’；
R6Ks2：5’-GTCAAAAATAAGTGCCTTCCGAATTCCCATGTCAGCCGTT-3’；
RP4a2：5’-ACCTGCAGGCATGCAAGCTTTCTGCTCTGATGCCGCATAG-3’；
tetS：5’-GGAAGGCACTTATTTTTGAC-3’；
tetA：5’-TCCTCAACGACAGGAGCACG-3’。
8. use of the plasmid combination of claim 1 for cloning large fragment DNA in pseudomonas putida genome.
9. A method for cloning large fragment DNA in pseudomonas putida genome using the plasmid combination of claim 1, comprising the steps of:
(1) Amplifying the front end and the rear end of a DNA region to be cloned respectively as homologous arms for upstream and downstream homologous exchange, respectively connecting the homologous arms into pBCPP51 and pBCPP52 cut by EcoRI, and transforming escherichia coli S17-1 lambda pir to obtain plasmids pBCPP51up and pBCPP52dn respectively;
(2) Introducing pBCPP51up into Pseudomonas putida through conjugal transfer, and integrating the position of an upstream homology arm in a genome through homologous exchange to obtain a primary integration strain with gentamicin resistance;
(3) Introducing pBCPP52dn into the primary integrated strain in the step (2) through conjugal transfer, and integrating the primary integrated strain into the genome through homologous exchange at the position of a downstream homology arm to obtain a secondary integrated strain with tetracycline resistance;
(4) Extracting genome of the secondary integrated strain, and selecting one enzyme cutting site in multiple cloning sites MCS on pBCPP51 and pBCPP52 for enzyme cutting, wherein the enzyme cutting site cannot exist on large fragment DNA to be cloned;
(5) The digested genomic fragment was subjected to ligation and circularization using T4 ligase, and E.coli was transformed, and a resistance plate containing gentamicin and tetracycline was coated, and the resulting plasmid contained pBBR1 oriV, mob, gentamicin resistance gene, large fragment DNA to be cloned, tetracycline resistance gene and pBBR1 rep on which pBBR1 oriV was replicated in this order.
10. The method of claim 9, wherein the E.coli in step (5) is E.coli XL10-Gold.
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