Promoter and carrier composed of same and application thereof
An improved promoter and a use thereof. An improvement is to mutate a nucleic acid sequence between −35 region and −10 region in a promoter region into recognition sites for an endonuclease. The improvement can overcome the problem that a strong promoter in a vector based on blue-white screening initiates the transcription or translation of foreign genes and a transcription or translation product might be toxic to a host and cannot be cloned, avoid the deficiency that frameshift mutation of a gene due to a lack of 1-2 bp of the vector at digestion sites results in false positive clones, and eliminate a false negative phenomenon that a plate is rich in blue spots due to a small fragment of foreign DNA and a reading frame of the gene which is unchanged by inserting the foreign DNA.
1 . A cloning vector, comprising:
a) a promoter comprising a −35 region, a −10 region, and one or more recognition sites for an endonuclease between the −35 region and the −10 region of the promoter, wherein:
i) a nucleic acid sequence between the −35 region and the −10 region is any one of SEQ ID NOs: 3-10; and
ii) the endonuclease is EcoRV, AleI, BamHI, XhoI or PmlI, or a combination thereof; and
b) a gene regulated by the promoter, wherein the gene is toxic to a host.
2 . The cloning vector of claim 1 , wherein the −35 region is not mutated.
3 . The cloning vector of claim 2 , wherein the gene toxic to the host is a lethal gene or a restriction enzyme gene.
4 . The cloning vector of claim 1 , wherein the gene toxic to the host is a lethal gene or a restriction enzyme gene.
5 . The cloning vector of claim 4 , wherein the lethal gene is a ccdB gene.
6 . The cloning vector of claim 5 , wherein a nucleic acid sequence of the ccdB gene is SEQ ID NO: 11.
7 . The cloning vector of claim 1 , wherein the vector is constructed from any one or a combination of at least two of pUC18, pUC19, pUC57, pCA, pCK, pCC and pCC1.
8 . The cloning vector of claim 1 , wherein the vector is linearized and has blunt ends at a recognition site for an endonuclease in the promoter.
9 . The cloning vector of claim 1 , wherein the vector further comprises a foreign DNA inserted into the vector at a recognition site for an endonuclease in the promoter.
10 . The cloning vector of claim 1 , wherein the vector further comprises a lacI expression element.
11 . A T vector comprising: the cloning vector of claim 1 , wherein the cloning vector is a linearized cloning vector and has blunt ends at a recognition site for an endonuclease in the promoter; and one added 3′ dideoxythymidine nucleotide at each end of the linearized cloning vector.
12 . A recombinant vector comprising a foreign DNA and the T vector of claim 11 , wherein the foreign DNA is ligated between the ends of the T vector.
13 . A method for preparing the T vector of claim 11 , comprising the following steps:
(1) replacing a lacZα gene on a vector with a gene toxic to a host, wherein the gene toxic to a host is regulated by an original promoter of a β-galactosidase;
(2) designing a primer for mutating a nucleic acid sequence between a −35 region and a −10 region in the original promoter to comprise one or more recognition sites for an endonuclease, and using the original promoter and a gene regulated by the original promoter as a template for PCR amplification, thereby producing a vector with a promoter comprising one or more recognition sites for an endonuclease, wherein the gene toxic to the host is regulated by the promoter comprising one or more recognition sites for an endonuclease;
(3) cyclizing the product in step (2) to obtain a cloning vector with the promoter comprising one or more recognition sites for an endonuclease; and
(4) linearizing the cloning vector in step (3), thereby producing blunt ends, and adding one 3′ dideoxythymidine nucleotide to each end of the linearized vector, to obtain the T vector.
14 . The method of claim 13 , wherein:
a nucleic acid sequence of the primer in step (2) is any one of SEQ ID NOs: 13-28;
the linearizing in step (4) is performed through endonuclease digestion and/or the PCR amplification;
the adding one 3′ dideoxythymidine nucleotide in step (4) is performed with a terminal transferase or a Taq DNA polymerase;
before step (1), the method further comprises performing codon optimization on the gene toxic to the host;
after step (1), the method further comprises inserting a foreign DNA into the vector;
step (3) is performed by a Gibson recombination method;
or a combination of the foregoing.
15 . A host cell, comprising the cloning vector of claim 1 , wherein the host cell is Escherichia coli.
16 . A method for cloning, comprising:
adding one A base to each 3′ end of a foreign DNA;
ligating the foreign DNA with added A bases to the T vector vector of claim 9 , thereby producing a recombinant vector;
introducing the recombinant vector into a host cell; and
cultivating the host cell under appropriate conditions, to obrain a positibe clone; wherein the host cell is Escherichia coli.
17 . A kit for cloning, comprising any one or a combination of at least two of:
(a) the cloning vector comprising the promoter of claim 1 ;
(b) a T vector comprising: the cloning vector of (a), wherein the cloning vector is linearized and has blunt ends at a recognition site for an endonuclease in the promoter; and one added 3′ dideoxythymidine nucleotide at each end of the linearized cloning vector;
(c) a recombinant vector comprising a foreign DNA and the T vector of (b); wherein the foreign DNA is ligated between the ends of the T vector; and
(d) a host cell comprising the vector of any one of (a) to (c).
18 . A cloning vector, comprising:
a) a promoter comprising a −35 region, a −10 region, and one or more recognition sites for an endonuclease between the −35 region and the −10 region of the promoter, wherein:
i) a nucleic acid sequence between the −35 region and the −10 region is any one of SEQ ID NOs: 3-10; and
ii) the endonuclease is EcoRV, AleI, BamHI, XhoI or PmlI, or a combination thereof;
b) a gene regulated by the promoter, is toxic to a host; and
c) a lacI expression element, wherein a nucleic acid sequence of the lacI expression element is SEQ ID NO: 12.