High copy number plasmids compatible with commonly used cloning vectors.
High copy number plasmids compatible with commonly used cloning vectors.
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DOI：
10.2144/00283bm02
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发表时间：
2000-03
期刊：
影响因子：
2.7
通讯作者：
Gregory J. Phillips;Sei-Kyoung Park;Damon Huber
中科院分区：
文献类型：
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作者：
Gregory J. Phillips;Sei-Kyoung Park;Damon Huber
A number of genetic and biotechnological applications are facilitated by the ability to transform Escherichia coli with more than one recombinant plasmid. For example, a plasmid that carries a gene of interest may require that a trans-acting regulatory protein be expressed from a second plasmid in the cell to effectively regulate its expression (9,16,29). In addition, purification of multisubunit complexes, or characterization of the interaction between gene products, can also be facilitated by elevated expression of the interactive components. In E. coli, for example, the Ffh protein interacts with a 4.5S RNA species to form a ribonucleoprotein complex known as the signal recognition particle (SRP) (17,18). To better understand the interactions between Ffh and 4.5S RNA we sought to jointly over-express both of the SRP components in E. coli. One strategy to do this was to individually clone ffh and ffs, which encode 4.5S RNA, into cloning vectors that are members of different incompatibility groups and transform both plasmids into the same E. coli strain. Some of the most commonly used plasmid vectors are derivatives of the ColE1-like plasmid pMB1, including pBR322 (2), and high copy number versions comprising the pUC (27,32) and pBluescript (Stratagene, La Jolla, CA, USA) family of vectors. Multicopy cloning vectors that are compatible with pMB1 plasmids include those that use the p15A origin of replication (ori) such as pACYC177, PACYC184 (4), as well as other derivatives (1). In general, these plasmids exist at a medium copy number of 15–20 plasmids/chromosome. Also described are cloning vectors derived from pSC101 (13,28) that are maintained at low copy number (6–8 copies/chromosome). These vectors permit construction of bacterial strains that harbor compatible plasmids. While medium to low copy number vectors may be suitable for many applications, their use can be limiting when high-level expression of a gene product is required. Therefore, to maximize expression of two different gene products we sought to construct new cloning vectors that are compatible with many commonly used plasmids and that replicate at a high copy number. Although replication of ColE1-like plasmids is dependent on DNA polymerase I and their replication is regulated by the interaction of two complementary RNA transcripts, distinct incompatibility groups have been identified (20,22). The ColE1-like plasmid RSF1030 (10), for example, is able to co-reside with both pMB1 and p15Aderivative plasmids, as well as with non-ColE1 vectors such as pSC101. The ori from RSF1030 was therefore selected for construction of a series of new cloning vectors. Wild-type RSF1030 is maintained at a copy number of 15–20 per chromosome equivalent, a copy number that is comparable to pBR322 (6), pACYC177 and pACYC184 (4). To construct a cloning vector that replicates at a high copy number yet maintains its compatibility with commonly used cloning vectors, a high copy number mutant of RSF1030 was isolated. An RSF1030 derivative, pST19 (22), was converted to a high copy number mutant by selecting transformants able to form colonies in the presence of elevated levels of ampicillin and methicillin. An overnight culture of DH5α transformed with pST19 was plated onto LB agar plates (21) containing 400 μg/mL ampicillin and 400 μg/mL methicillin (31). Several colonies that arose under these selective conditions were screened using a rapid cell lysis procedure (19) to identify transformants that contained elevated quantities of plasmid DNA. A single mutant that yielded significantly more plasmid DNA than cells transformed with pST19 was selected for further analysis. DNA sequence analysis of a 1.5 kb segment containing the ori from the high copy number mutant revealed a single base pair alteration located in a region that encodes the RNAII replication primer and the antisense RNAI transcript (Figure 1A). Although this mutation does not appear to have been previously reported in selections for Benchmarks
