Construction of new vectors for cloning of promoters

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.     

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.     

Construction of shuttle vectors for cloning inVibrio cholerae andEscherichia coli

Starting from a naturally occurring cryptic plasmid pVC540 ofVibrio cholerae non-OI. strain 1095, a number of plasmid vectors have been constructed for cloning genes inVibrio cholerae by introducing antibiotic resistance markers containing a set of unique cloning sites. The constructs pVC810 and pVE920 have the origins of bothVibrio cholerae andEscherichia coli replicons and are stable in both organisms in the absence of selective pressure. These plasmids can serve as shuttle vectors betweenEscherichia coli andVibrio cholerae. The plasmid vectors reported here along with the demonstration of transformation inVibrio cholerae by plasmid DNA will facilitate genetic analysis of this important human pathogen.     

Construction of shuttle vectors for cloning inEscherichia coli andAcetobacter pasteurianus

New cloning vectors were prepared with the aid of a large plasmid isolated fromAcetobacter pasteurianus and from plasmids pBR322 and pUC4-KAPA. Of the prepared cloning vectors, pACK5 contains a gene coding for kanamycin resistance, pACT7 and pACT71 contain a gene coding for tetracycline resistance and vector pACG3 with a gene coding for both kanamycin and tetracycline resistance. The vectors prepared only contained the beginning of replication from the pAC1 plasmid and possessed the ability to replicate withinE. coli andA. pasteurianus. The vectors are highly stable in both strains and during the 5-d cultivation under nonselective conditions are not eliminated.     

Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors

pSSU1, a native plasmid of Streptococcus suis DAT1, was used to construct pSET-series shuttle vectors. In addition to the replication function of pSSU1, these vectors contain the multiple cloning sites and lacZ' gene from pUC19, which means that X-gal screening can be used to select recombinants in Escherichia coli. pSET1, pSET2, and pSET3 carry cat, spc, and both of these genes, respectively, as selectable markers. These vectors could be introduced into S. suis, E. coli, Salmonella typhimurium, S. pneumoniae, and S. equi ssp. equi by electrotransformation. The recA gene was cloned from S. suis and sequenced, and this information was used in the construction of a recA mutant of S. suis. Transformation frequencies and/or plasmid stability of all pSET vectors tested were decreased in both S. suis and E. coli recA mutants compared with the parental strains. These results suggested that functional RecA protein improved the maintenance of pSET vectors in both S. suis and E. coli. Moreover, cloning of the functional S. suis recA gene into pSET2 and complementation analysis of the recA mutant were successful in S. suis but not in E. coli. These results showed that pSET vectors are useful tools for cloning and analyzing S. suis genes in S. suis strains directly.     

Construction and characterization of new coliphage M13 cloning vectors

New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF). These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII. Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene. These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations. Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors. Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand. Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies. Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe. Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert.     

Construction and use of integrative vectors to express foreign genes in mycobacteria

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide’vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18kDa gene and expression in M. smegmatis was obtained.     

Construction of linear plasmid vectors for cloning in Escherichia coli cells]

Prophage of the temperate coliphage N15 is a linear plasmid with covalently closed ends. The central part of plasmid N15 genome responsible for vegetative phage growth was replaced by DNA fragments containing genes for selective markers which have unique restriction sites. As a result a family of linear plasmid vectors was constructed. Their size is about 20 kb and their capacity is comparable with that of cosmid vectors.     

Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors.

We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Novel cloning vectors for Bacillus thuringiensis

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Secretion cloning vectors in Escherichia coli

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-III. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindIII or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoter-operator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of beta-lactamase was inserted into the EcoRI site. Upon induction of gene expression, beta-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of beta-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, beta-lactamase was accumulated to 20% of total cellular protein without any detectable accumulation of pro-beta-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, beta-lactamase with the authentic NH2-terminal sequence was produced, in even larger amounts than the beta-lactamase with the linker sequence.     

Structurally stable Bacillus subtilis cloning vectors

Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does. In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.     

Versatile mercury-resistant cloning and expression vectors

Cloning vectors have been constructed employing two diverse replicons, IncQ and P15A. Both vectors confer resistance to kanamycin (Km) and mercuric ions (Hg2+). One of these vectors, pDG105, is a broad-host-range, nonconjugative, oligocopy IncQ plasmid, which is capable of transforming Escherichia coli, Acinetobacter calcoaceticus, and Pseudomonas putida. The second vector, pDG106, is a narrow-host-range, multicopy cloning vector compatible with pBR322. Both vectors contain unique cloning sites in the Km-resistance gene for HindIII, SmaI, and XhoI, as well as unique EcoRI and ScaI sites in the mer operon. Cloning into the EcoRI site in the mer operon results in the mercury "supersensitive" phenotype, easily detectable by replica plating. Insertion of the galK gene into the EcoRI site in the mer operon results in Hg2+-inducible galactokinase activity, demonstrating the application of these plasmids as regulated expression vectors.     

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

Background  

The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.