Phasmids: hybrids between ColE1 plasmids and E. coli bacteriophage lambda

Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda. The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombinants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.     

A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd

DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.     

Synthesis of a 33-member polynucleotide containing the "core" att site of phage lambda DNA and its cloning

Two polynucleotides containing 33 monomeric units were synthesized by a solid-phase phosphotriester method. These polynucleotides form a duplex with protruding 5'-ends, which allows to clone the duplex in EcoRI site of a cloning vehicle. Each polynucleotide was purified by electrophoresis in polyacrylamide gel, and the duplex obtained was cloned in EcoRI site of pUR 222 plasmid DNA. The structure of the cloned duplex containing the "core" att site of phage lambda was confirmed by sequencing.     

The DNA between Rz and cosR in bacteriophage lambda is nonessential

Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda. The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.     

The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.     

New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome.

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage lambda attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the lambda attachment site attP and (ii) a helper plasmid, bearing the lambda int gene, transcribed from the lambda PR promoter under the control of the temperature-sensitive repressor cI857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 degrees C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 degrees C. These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.     

Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene

The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E. coli lacZ gene is reported. A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980). Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity. The E. coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids. The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.     

Plasmids supplying the Q-qut-controlled gam function permit growth of lambda red- gam- (Fec-) bacteriophages on recA- hosts

A plasmid, pgam, has been constructed which expresses the phage lambda gene, gam, under the control of the lambda late promoter, p'R, contained in a form of a p'R-qut-t'R1 module. Lambda red- gam-, which normally do not grow on recA- hosts, are able to grow on recA- hosts containing pgam, because their Q function can turn on the gam gene expression. This facilitates cloning with lambda red- gam- vectors in recA- hosts.     

Transactivation of p'R promoter of phage lambda

The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated. The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene. The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA. The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322.     

Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element ( att  P) and the chromosomal ( att  B) sites. These regions share an identity segment of 58 bp extending from the anti-codon loop through the 3' end of a tRNA^Pro gene. To facilitate the study of the att  B site, the int and xis genes, expressed from an inducible promoter, and att  P from pSAM2 were cloned on plasmids in Escherichia coli . Compatible plasmids carrying the different att  B regions to be tested were introduced in these E . coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in att  B for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of att  B; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long att  B with att  P, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.     

Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.     

A universal instinct for designing thermoregulated promotors in gram-positive bacteria]

The construction of plasmid pVKH300, which is useful for modifying any promoter into the thermoregulated form in B. subtilis cells, is presented. The main features of the plasmid are the presence of effectively expressed in B. subtilis lambda C1857 gene and recognition site of BglII restriction enzyme between OR2 and OR3 lambda phage operator sites. Promoterless alpha-amylase gene of B. amyloliquefaciens is used as a reporter gene for promoter cloning into BglII site of pVKH300. Examples of promoter-containing DNA fragments cloning with pVKH300 as vector are presented. It was found that the best regulated promoter, in a plasmid named pVKH332, was cloned in such a way that the distance between central nucleotides of OR2 and OR3 is equal to integer number of DNA helix turns (84 b.p. in the case).     

Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter

By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts.