Boundaries of the Origin of Replication: Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replication (ori); pET-28a(+) contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+) replicon–comprising the ori, RNAI, RNAII, and Rom–with the p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. This essential need is currently not filled.     

共表达TrxA和DsbC对富含二硫键的异源蛋白在大肠杆菌中表达的影响

以复制子为p15A的质粒pACU184为基础 ,构建了 3种表达硫氧还蛋白 (TrxA)或 和二硫键异构酶 (DsbC)的表达质粒 .经IPTG诱导 ,克隆的DsbC和TrxA都以可溶的形式高表达 .分别将构建的 3种表达质粒与复制子为colE1并克隆有人源化鼠抗人纤维蛋白单链抗体 低分子量尿激酶融合基因 (C6 UK)的表达质粒共转化大肠杆菌XL1 blue ,在 30℃用IPTG诱导表达 .SDS PAGE显示 ,共表达TrxA或DsbC都能导致C6 UK融合蛋白的部分可溶性表达 ,而且同时共表达TrxA和DsbC 2种分子时 ,C6 UK完全以可溶形式表达 ,但表达量降低 .分别用溶圈法和ELISA检测了各种共表达时可溶表达产物的生物活性 .结果显示 ,只有共表达DsbC时才能检测到明显的C6 UK融合蛋白的双功能     

A novel high-copy plasmid, pEC, compatible with commonly used Escherichia coli cloning and expression vectors

A 66164-bp cryptic plasmid, pEIB1, was isolated from strain Vibrio anguillarum MVM425 and sequenced. A plasmid carrying a 1089-bp fragment, containing the minimal replication region of pEIB1, a kanamycin-resistance marker and an l-arabinose promoter, designated pEC, was maintained as a high copy plasmid in E. coli and stably inherited in the absence of antibiotic selection. Significantly, pEC was compatible with the widely used ColE1, pSC101 and p15A replicons making it a useful tool for a dual-plasmid expression system.     

New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; a potential vector for a two-plasmid expression system

We constructed pIGPZ, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region, and a promoter for constitutive expression of cloned genes. pIGPZ has the same high level of stability as the original plasmid, even in the absence of antibiotic selection. Furthermore, we show that pIGPZ is compatible with ColE1-based plasmids and a pSC101-like plasmid. All the characteristic elements of theta-replicating plasmids were found in the pIGPZ putative origin of replication. Finally, we demonstrate that pIGPZ can be used in a double-plasmid expression system by co-expressing UBP1 protease from pIGPZ with ubi-interferon alpha (IFNA13; GenBank Accession No. NM_006900.3) or ubi-human growth hormone (ubi-hGH; patent No. WO 2005/066208 A2) cloned in another plasmid. In this system, both ubi-interferon alpha and ubi-human growth hormone were deubiquitinated efficiently in E. coli cells.     

Localization of genes responsible for replication and immunity to colicin A on plasmid ColA-CA31

The region containing the origin and regulatory sites for replication as well as the immunity gene (iaa) have been localized on the plasmid ColA-CA31. The region involved in replication functions of ColA can be hybridized with that of ColE1. It is located between 1 and 1 kb on the plasmid map previously published (Morlon et al. 1982a). A 0.50 Kb HincII fragment of ColA can be weakly hybridized to the ColE1 immunity region. This fragment contains iaa since directed in vitro mutagenesis at an internal restriction site can abolish the immunity to colicin A; however, it does not contain the entire iaa. Knowing the localization of regions involved in autonomous DNA replication and immunity, a mini-ColA plasmid was constructed that contains these two regions. The mini-ColA of 2.8 Kb can be amplified in the presence of chloramphenicol and confers the immunity to transformants. It thus constitutes a useful cloning vector. Expression of ColA and of the various constructed plasmids in the maxicell system suggests that the immunity protein has a molecular weight of about 18-20 Kd.     

Influence of fd gene 2-protein and the viral replication origin on the compatibility of pfd-plasmids.

Plasmids with the replication origin of bacteriophage fd, the pfd-plasmids, were investigated for compatibility in E. coli cells expressing fd gene 2-protein. This was measured by transformation of Ca-treated cells with and without a residing pfd-plasmid. When the two plasmids contained the complete intergenic region of bacteriophage fd, they were fully compatible in contrast to the situation in which at least one plasmid had a shortened origin for viral strand replication. This incompatibility effect was partially compensated for by a pfd-plasmid with a short origin and with the fd gene 2. The fd replication origin on a colEl plasmid did not affect compatibility in polA+ cells indicating its idling in the presence of the colEl origin. It can be concluded that a short replication origin requires high amounts of gene 2-protein in contrast to the long origin. Accumulation of replication intermediates severely interferes with host cell metabolism.     

Construction of a shuttle vector for use in Francisella tularensis

Abstract The characterisation of virulence factors of Francisella tularensis has been hampered by the lack of genetic system for the bacterium. In this study, a shuttle vector was constructed that can replicate autonomously in F. tularensis and Escherichia coli . To obtain this vector, the p15A replication origin of E. coli plasmid pACYC184 was introduced into a plasmid derivative of plasmid pFNL200, a plasmid which only can replicate in F. tularensis . The resulting shuttle vector, designated pKK202, harboured resistance genes for chloramphenicol and tetracycline. This vector might be used as a basis for the studies of virulence factors of F. tularensis .     

An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1

We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome

Summary A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment.The cloned plasmid pMS102-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102-B7 was less than 1/1,000 of that by the vector plasmid pMS102. The number of copies of pMS102-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts.Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.     

Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells.

The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.     

A set of plasmids constitutively producing different RNA levels in Escherichia coli.

New plasmids were developed for the in vivo expression of RNA in Escherichia coli. These plasmids combine constitutive promoters of different strengths with different origins of replication to provide a 75-fold range of expression of amber suppressor tRNA. The plasmids are either pMB1, p15A or temperature-sensitive SC101 replicons, and can be used in two plasmid systems for studying RNA-protein interactions. The temperature-sensitive SC101 plasmids may be useful as gene replacement vectors. Another vector that is suitable for generating lethal mutations was constructed in a plasmid containing a regulatable phage T7 promoter.     

Complete sequence of virulence plasmid pEIB1 from the marine fish pathogen Vibrio anguillarum strain MVM425 and location of its replication region

AIMS: The aim of this study was to determine the whole DNA sequence of pEIB1, one pJM1-like virulence plasmid from Vibrio anguillarum MVM425 and locate the replication region. METHODS AND RESULTS: DNA sequence of virulence plasmid pEIB1 from V. anguillarum MVM425 was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking. The whole nucleotide sequence of pEIB1 comprises 66,164 bp, encoding 44 open reading frames (>400 bp) containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. With no demonstrated replication origin, the Sau3AI partial digested plasmid DNA fragments of pEIB1 were ligated into the BamHI-fragment containing the kanamycin-resistance gene (Kmr). For there is no effective transformation in V. anguillarum, the ligated DNA was first introduced into E. coli JM83, and the transfomants were selected for resistance to kanamycin. It was demonstrated with southern blotting and DNA sequencing that plasmid pEIB7 containing the Sau3AI DNA fragment of pEIB1 (from 12516 to 13957) has the ability to replicate in E. coli JM83 and V. anguillarum MVM425sh. The segregational stability of plasmid pEIB7 kept in 100 and 4% in E. coli JM83 and V. anguillarum MVM425sh respectively when the cells were cultured in 200th generation. In following experiments, we also found that plasmid pEIB7 replicated at a middle-copy number of 10-40 in JM83, while at a high-copy number of 100-300 in MVM425sh. Moreover, pEIB7 can survive in V. alginolyticus, another fish pathogenic. CONCLUSIONS: With the whole DNA sequence of pEIB1 determining, it was found that pEIB1 showed microheterogeneity in its restriction endonuclease patterns with pJM1 though their DNA sequences had slight difference. According to the complete DNA sequence of pEIB1, its replication region was located from 12516 to 13957. And this replication region is compatible to pUC18 (pMB1), pKA3 (pSC101) and p15A: caiE (p15A). SIGNIFICANCE AND IMPACT OF THE STUDY: The worldwide vibriosis marine pathogen V. anguillarum strains contain common virulence, pJM1-like plasmids, independent on the geographical source. The pEIB1 was the second common virulence plasmid, which sequence was determined. Its sequence is highly homologous to pJM1 as they both encode biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. Some interesting features as in pJM1 were also identified, such as transposon-like structures. So it can be deferred that the whole DNA sequences of virulent plasmid pEIB1 will be great helpful to future revealing these V. anguillarum virulence-related genes derived during evolution from transposition events or horizontal transfer of genes potentially originating in other organisms. Another result, replication region of pEIB1 locating is the first report about replication of pJM1-like plasmid. This work will be useful for researching pJM1-like plasmid replication mechanism in V. anguillarum.     

Replication of a plasmid containing two origins of bacteriophage f1

A chimeric plasmid was constructed that contains a tandem duplication of the bacteriophage f1 origin of DNA replication. This plasmid replicates stably in the absence of phage. When cells carrying this plasmid are infected with f1, two new plasmid-derived DNA species are generated: a smaller, chimeric plasmid containing only one f1 origin of replication, and a miniphage, the genome of which consists of the f1 fragment that was located between the two f1 origins of the original plasmid. These data support the hypothesis (Horiuchi, 1980) that the nucleotide sequence recognized for initiation of plus strand synthesis in f1 DNA replication is also the signal for its termination.     

Expression of incompatibility by derivatives of the broad host-range inc P-1 plasmid RK2.

Summary A segment of DNA encoding incompatibility on the inc P-1 plasmid pRK248 was identified by the analysis of deletions generated in vitro, and then cloned into several unrelated and mutually compatible plasmids. These derivatives were tested for expression of P-1 incompatibility. It was demonstrated by transformation experiments that P-1 plasmids were efficiently eliminated from an E. coli host following introduction of any one of the derivatives. However, all the derivatives were compatible with each other. The cloned segment of pRK248 DNA is itself capable of autonomous replication, without being cloned into any plasmid, if plasmid-specified gene products are provided in trans. This satellite plasmid is eliminated from the cell by the inc P-1 plasmid pRK286. The results argue against a partitioning mechanism as the basis for P-1 incompatibility but are consistent with incompatibility being the consequence of negative regulation of copy number. For the inc P-1 system, susceptibility of the plasmid to elimination, but not its ability to eliminate, requires that the P-1 replication system is active.     

Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system

High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or “leaky” production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacI^q gene have been constructed to permit titration of the LacI protein. When a high-copy plasmid is transformed along with the appropriate lacI^q-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression.     

Construction of a food-grade multiple-copy integration system for Lactococcus lactis

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori⁺) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori⁺ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-cross-over integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5–15) × 10⁻² copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest. Received: 23 September 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997