Positive-selection Cloning Vectors Research Articles

Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli

Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.4 Bacillus pumilus lipase gene whose expressed protein is toxic and lethal to Escherichia coli JM109 (DE3) cells. The determinant of toxicity was identified through Error-prone PCR to be the nature of amino acid residue resident at position 28 of the mature lipase protein. The presence of Thr/Ser28 within the mature lipases of B. pumilus and B. licheniformis resulted in lethality to E. coli cells. However, the Thr28Ala or Thr28Gly mutations relieved the lethal phenotype of mature Family I.4 Bacillus lipases. The toxic effect of the expressed mature B. pumilus lipase protein was exploited in the development of a positive selection cloning vector. The B. pumilus lipase gene was synthesised to contain 13 unique silent restriction sites within the ORF, and placed under the regulation of T7 promoter of the pET expression system. Insertional inactivation of the gene's toxic protein was achieved by cloning DNA fragments of different sizes within the designed multiple cloning sites. The toxic effect of the lipase protein was disrupted indicating the potential of the gene for application in suicidal positive selection cloning vectors. The results revealed that protein expression and engineering studies aimed at optimal production of mature Family I.4 Bacillus lipases in E. coli should take into consideration the nature of amino acid 28 resident.

PKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli.

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.

Positive Selection, Cloning Vectors for Gram-Positive Bacteria Based on a Restriction Endonuclease Cassette

Lactococcus lactiscontains numerous restriction and modification (R/M) systems of different specificities. A novel IIS type R/M system encoded by theLlaI operon has previously been characterized from theL. lactisconjugative plasmid pTR2030. TheLlaI operon is composed of six genes: First, a small regulatory genellaICprecedes the methylase genellaIM.The following three genes,llaI.1, llaI.2, llaI.3,are all essential for restriction endonuclease activity and are designed as the restriction cassettellaIR.The forth open reading frame of unknown function follows thellaIRgene cassette. We have successfully subcloned the threellaIRgenes,llaI.1, llaI.2,andllaI.3,withoutllaIM,as a suicide cassette into the three shuttle vectors pTRKL2, pTRKH2, and pBV5030. A promoter (P6) fromLactobacillus acidophilusATCC4356, which is functional inE. coli,lactococci, and lactobacilli (Djordjevic and Topisirovic, unpublished) was cloned upstream of the three gene cassette. Restriction activity was evaluated inEscherichia coliand several gram-positive bacteria. ThellaIRrestriction cassette was not functional inE. coli,but its presence was lethal toL. lactis, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Carnobacterium pisicola, Enterococcus faecalis, Bacillus subtilis,andLeuconostoc gelidum.Several novel, positive selection cloning vectors were developed that can exploit unique cloning sites within thellaIRcassette. Insertions inllaI.1resulted in complete inactivation of restriction activity and provided unconditional selection for recombinant plasmids in surviving transformants. These positive selection cloning vectors are the first for gram-positive bacteria that are based on a restriction endonuclease cassette. Functional activity of thellaIRgenes in various gram-positive bacteria would also enable use of these cloning vectors for positive selection of promoters, terminators, and regulatory sequences across these genera.

New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers

Recently, we described pKIL18/19 positive-selection cloning vectors which rely on the inactivation of the cytotoxic ccdB gene [Bernard et al., Gene 148 (1994) 71-74]. They are new tools for simplifying gene cloning/sequencing procedures, as only recombinant DNA allow the formation of viable colonies. To enhance positive-selection capabilities, putative translational reinitiation codons located within the pKIL18/19 ccdB were modified by site-directed mutagenesis. New pKIL-derived vectors with kanamycin (Km R) or chloramphenicol (Cm R) resistance-encoding genes were also constructed. In addition, a new host derived from the DH2 strain was developed.

Positive-selection vectors using the F plasmid ccdB killer gene

Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter. They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites. When an Escherichia coli wild-type gyrA + strain is transformed by such vectors, the ccdB gene product blocks bacterial growth. However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E. coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies. The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB [Bernard and Couturier, J. Mol. Biol. 226 (1992) 735–745]. Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host. Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.

Generalized and mobilizable positive-selection cloning vectors

We have modified the positive-selection cloning vector pUN121 to expand the numbers of unique cloning sites by insertion of a multiple cloning site into the λ, ficI gene without disrupting its repressor function, resulting in plasmid pSKM10. Plasmid pSKM10 has seven restriction enzyme sites suitable for general cloning purposes. A mobilizable version (pSKM11) of pSKM10 was constructed by insertion of the IncP mob sequence which permitted mobilization of the plasmid into a wide variety of Gram − bacteria.

Establishment of a genomic bank of bovine herpesvirus 1 using a novel positive selection plasmid vector

A small positive selection cloning vector, designated pSiigl, suitable for the construction of genomic banks in E. coli is described and used for the establishment of a bank of the bovine herpesvirus 1 genome. Hybrid transformants are directly selected on agar plates containing ampicillin. The vector is based on the replicon of Rl and has a lambda P R promotor inserted upstream of the replication control genes. The vector has an uncontrolled (runaway) replication and is lethal to the host cell unless the P R promotor is brought under trans-acting control of the lambda cI repressor or runaway replication is blocked by an insertion between the P R promotor and the replicon. The vector contains a unique BglII site between P R and the replicon which is suitable for insertion of genomic DNA.

A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers

Several families of positive-selection cloning vectors were constructed, based on the principle of palindrome nonviability first used by Hagan and Warren [Gene 19 (1982) 147–151]. Each vector, derived from either pBR322 or RSF1010 (a broad-host-range plasmid), contains a long inverted repeat (2 × 366 to 2 × 1008 bp) ending in a symmetrical polylinker. Plasmids with long palindromes are not viable in most strains of Escherichia coli and in at least one Gram-positive bacterium. These palindrome-containing vectors therefore transform such strains at a very low frequency unless a DNA fragment is cloned within the polylinker at the center of the palindrome. Transformation by plasmids lacking an insert is reduced by two to four orders of magnitude. Such vectors can be propagated in a palindrome-tolerant strain; however, long symmetrical deletions then occur within the palindrome. To suppress the resulting deletion derivatives, vectors have been constructed so that an extensive deletion would remove the selectable marker. Alternatively, the vectors can be propagated in any strain of E. coli so long as the palindrome is interrupted by a nonpalindromic DNA fragment. We also present several symmetrical polylinkers and drug-resistance cassettes within the vectors. These components can be interchanged to make new positive-selection vectors as needed, and the cassettes are useful in insertional mutagenesis as well. A general method is described to convert virtually any small or medium-sized plasmid into a positive-selection vector.

Positive selection vectors based on xylose utilization suppression

High levels of xylose isomerase activity in wild-type Escherichia coli strains results in a Xyl − phenotype. This phenomenon was exploited for the development of a versatile positive selection system. The xylA promoter was deleted with the exonuclease BAL 31 and the resulting structural gene was inserted into the SmaI site ofpUC9, yielding the prototype vector, pLX100. In this construct xylA expression is placed under the transcriptional control of the lac promoter. Transformation of any wild-type E. coli strain with pLX100 results in high levels of xylose isomerase and a Xyl − phenotype. Decreasing the activity below a critical level (approx. 100 u) restores the Xyl + phenotype. pLX100 contains contiguous restriction sites for HindIII, PstI, BamHI and XhoI, suitable for positive selection cloning experiments. E. coli transformants containing pLX100 cannot grow in minimal medium with xylose unless a DNA fragment is inserted into any one of the unique restriction sites. This makes the plasmid an ideal positive-selection cloning vector.

Expression of Streptococcus sanguis antigens in Escherichia coli: cloning of a structural gene for adhesion fimbriae.

Chromosomal DNA from Streptococcus sanguis FW213 was partially digested with EcoRI and ligated into the positive-selection cloning vector pOP203(A2+). The ligation mixture was used to transform Escherichia coli K-12, and 4,500 transformants were examined. The tetracycline-resistant colonies had inserts averaging 3.2 kilobases. The entire colony bank was screened by colony immunoassay with polyclonal rabbit serum raised against S. sanguis FW213 whole cells. Thirty recombinant colonies produced stable positive reactions of various intensities, indicating that S. sanguis antigens could be expressed in E. coli. Restriction endonuclease digestion of these clones suggested that 26 of the clones were unique. Only two clones, VT616 and VT618, gave positive reactions with fimbria-specific antisera. That the gene coding for the antigen was located on the plasmid was confirmed by demonstrating that the presence of the plasmid was linked to antigen production. Western immunoblot analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that both clones produced a fimbrial peptide of Mr 30,000. The two recombinant plasmids were shown by Southern analysis and restriction mapping to contain the same 6-kilobase EcoRI fragment inserted in opposite orientations. Southern hybridization confirmed that this fragment is present in S. sanguis genomic DNA. The Mr 30,000 protein gene was expressed in both orientations, suggesting that the fimbrial promoter is located on the 6-kilobase fragment. These results show that at least one streptococcal fimbrial gene can be cloned and expressed in E. coli.

A novel plasmid vector allowing positive selection for cloned fragments

The use of plasmid pMM102 as a positive selection cloning vector is described. This plasmid is derived from pBR322 and contains the DNA encoding microcin B17 production and immunity. RecA−Escherichia coli K12 cells containing this plasmid are unable to grow in minimal medium. Inactivation of any of the 4 genes required for microcin production allows the bacterial host to produce colonies. This property has been used to clone DNA inserts in the unique sites for restriction endonucleases BglII, SacI, SacII and SmaI in pMM102. DNA fragments with asymmetrical termini of many different kinds can also be cloned. We have also identified a fragment in the wild-type plasmid pMccB17 that suppresses the pMM102-induced growth inhibition.

A novel plasmid vector allowing positive selection for cloned fragments

The use of plasmid pMM102 as a positive selection cloning vector is described. This plasmid is derived from pBR322 and contains the DNA encoding microcin B17 production and immunity. RecA−Escherichia coli K12 cells containing this plasmid are unable to grow in minimal medium. Inactivation of any of the 4 genes required for microcin production allows the bacterial host to produce colonies. This property has been used to clone DNA inserts in the unique sites for restriction endonucleases BglII, SacI, SacII and SmaI in pMM102. DNA fragments with asymmetrical termini of many different kinds can also be cloned. We have also identified a fragment in the wild-type plasmid pMccB17 that suppresses the pMM102-induced growth inhibition.

Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectors

The genes encoding the EcoRI restriction-modification (R/M) system have been separately cloned onto compatible plasmids. We have shown that the EcoRI restriction gene is expressed in the total absence of methylase enzyme and confirmed that a temperature-sensitive mutant is defective in EcoRI modification activity at higher temperatures. Insertion of transcriptional terminators into the restriction gene had no detectable effect on EcoRI modification activity. This strongly suggests that a separate promoter exists for the methylase gene. Analysis of the published sequence shows that the methylase gene promoter may overlap with the COOH-terminal region of the endonuclease structural gene. The temperature-sensitive EcoRI system has been exploited in the construction of two plasmid cloning vectors, pLV57 and pLV59, which can be used to select positively for transformants bearing recombinant plasmids; cloning of a DNA fragment into pLV57 or pLV59 at the unique HindIII, BglII, or PstI sites inactivates the EcoRI restriction gene and permits the hybrid plasmid to survive at 37°C. The temperature-sensitive modification activity of these vectors should also facilitate the introduction of EcoRI linkers into DNA cloned in this way.