(1) The pBR322 plasmid is a 4362 bp circular double-stranded DNA vector with two drug resistance genes (tetracycline and ampicillin), one origin of replication and a plurality of restriction enzyme cleavage sites for cloning. When Escherichia coli lacking the resistance gene was successfully transformed by pBR322, it obtained antibiotic resistance from the plasmid. Both antibiotic genes contain different single cleavage sites for insertion of foreign DNA. Generally, only one antibiotic gene is selected for insertion of foreign DNA. The antibiotic resistance is inactivated after exogenous DNA insertion. Another antibiotic resistance gene is used as a positive clone for screening for transformed bacteria.
(2) The pUC series plasmid is a 2.7Kb double-stranded DNA plasmid with an origin of replication, an ampicillin resistance gene and a multiple cloning site, and the multiple cloning site is expressed in the LacZ gene product-β-galactosidase. The amino-terminal fragment, when the LacZ gene-mutated Escherichia coli strain (M15) was transformed with the pUC plasmid, the ability to decompose galactose was restored because the α-peptide expressed by the plasmid supplemented the α-peptide which was lost in Escherichia coli. Blue clones were grown on medium supplemented with IPTG and X-gal. If foreign DNA is inserted into the multiple cloning site, since it destroys the expression of the α-peptide, the blue clone cannot be grown in the medium to which IPTG and X-gal are added, which is called blue-white screening.
Second, single-strand filamentous phage and phagemid
(1) E. coli filamentous phage including M13 phage f phage, etc., the genome of which is a single-strand closed-loop DNA molecule, wherein the M13mp series cloning vector is a vector modified with wild type M13, inserted with a multiple cloning site and a LacZ gene. Therefore, IPTG and X-gal can also be used for blue-white screening. After M13 is infected with Escherichia coli, it is converted into double-stranded DNA by using infectious single-stranded DNA (positive strand) as a template under the action of enzymes in the bacteria. It is called replica DNA (RF DNA). Generally, when there are 100-200 copies of RF DNA in each cell, replication is stopped, producing an infectious intact single-stranded filamentous phage and secreting it away from the cells. Escherichia coli infected with M13 can continue to grow without lysis, but the growth rate is slower than that of normal bacteria. The bacterial culture medium infected with M13 can be centrifuged to extract RF DNA from the cells for molecular cloning operations such as restriction enzyme cleavage. For use, from the supernatant after centrifugation, phage particles can be precipitated by polyethylene glycol (PEG), single-stranded DNA (ss DNA) can be extracted for DNA sequence analysis, and in vitro localization mutation can be used.
(2) Phagemid Inserts a copy of the single-stranded phage replication initiation site DNA into the plasmid DNA, which constitutes a phagemid, such as pGEM-3Zf(-), which is a phagemid. The phagemid can be manipulated like a normal plasmid, but when it is necessary to prepare a single-stranded DNA, it is necessary to add a helper phage at the time of culture, and the commonly used helper phage has M13KO7 and R408. After the cultured bacteria solution is centrifuged to remove the cells, PEG is added to the supernatant to precipitate particles containing single-stranded DNA. Phagens are also commonly used for DNA sequence analysis and in vitro localization mutations. In addition to the above two types, the commonly used vectors for molecular cloning include phage and cosmid. For details, please refer to the relevant data.
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