Cloning Cassette Research Articles

CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system is being used successfully for efficient and targeted genome editing in various organisms, including the nematode Caenorhabditis elegans. Recent studies have developed a variety of CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: nonhomologous end joining and homologous recombination. Here, we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning (canonical marker-free and cassette selection methods), as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the germline. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Guide RNA preparation Alternate Protocol 1: sgRNA cloning using fusion PCR Basic Protocol 2: Preparation of a repair template for homologous recombination Alternate Protocol 2: Preparation of repair template donors for the cassette selection method Basic Protocol 3: Injecting animals Basic Protocol 4: Screening transgenic worms with marker-free method Alternate Protocol 3: Screening transgenic worms with cassette selection method.

A zero-background CRISPR binary vector system for construction of sgRNA libraries in plant functional genomics applications

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing is a ground-breaking biotechnology for agricultural applications such as precision breeding in crop plants. Agrobacterium-mediated CRISPR delivery has been successfully adapted for gene knockout applications for basic research and agricultural technology development. However, selecting proper single-guide RNA (sgRNA) for CRISPR binary constructs to induce double-strand break in certain target genes has presented difficulties mainly due to unpredictable in vivo sgRNA activities. Therefore, more than three independent CRISPR constructs, each harboring different sgRNAs, are often applied to ensure the desired CRISPR-induced knockout alleles. Here, we report a zero-background CRISPR binary vector platform, featuring ccdB conjugation within sgRNA cloning cassette, which is later removed by AarI endonuclease, that allows positive survival selection for bona-fide sgRNA clones and effective exclusion of uncut or self-ligated ‘background’ negative clones. We demonstrate the advantage of using the zero-background CRISPR binary platform in a high-throughput pooled cloning strategy of multiple different sgRNAs which produced Agrobacteria containing multiple sgRNAs without any background. We also tested the integrity of pooled CRISPR sgRNA construct libraries during extended bacterial culture and during the transfer between Escherichia coli to Agrobacterium, and verified that the fidelity of sgRNA species representation was faithfully maintained during library generation.

Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit.

SummaryKiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.

Flexible and rapid construction of viral chimeras applied to hepatitis C virus.

A novel and broadly applicable strategy combining site-directed mutagenesis and DNA assembly for constructing seamless viral chimeras is described using hepatitis C virus (HCV) as an exemplar. Full-length HCV genomic cloning cassettes, which contained flexibly situated restriction endonuclease sites, were prepared via a single, site-directed mutagenesis reaction and digested to receive PCR-amplified virus envelope genes by In-Fusion cloning. Using this method, we were able to construct gene-shuttle cassettes for generation of cell culture-infectious JFH-1-based chimeras containing genotype 1–3 E1E2 genes. Importantly, using this method we also show that E1E2 clones that were not able to support cell entry in the HCV pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system. This method can be easily applied to other genes of study and other viruses and, as such, will greatly simplify reverse genetics studies of variable viruses.

Self-Cloning CRISPR.

CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc.

Cloning-free CRISPR

SummaryWe present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.

Tools for Targeted Genome Engineering of EstablishedDrosophilaCell Lines

We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies.

Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

The ability to rapidly customize an expression vector of choice is a valuable tool for any researcher involved in high-throughput molecular cloning for protein overexpression. Unfortunately, it is common practice to amend or neglect protein targets if the gene that encodes the protein of interest is incompatible with the multiple-cloning region of a preferred expression vector. To address this issue, a method was developed to quickly exchange the multiple-cloning region of the popular expression plasmid pET-28 with a ligation-independent cloning cassette, generating pGAY-28. This cassette contains dual inverted restriction sites that reduce false positive clones by generating a linearized plasmid incapable of self-annealing after a single restriction-enzyme digest. We also establish that progressively cooling the vector and insert leads to a significant increase in ligation-independent transformation efficiency, demonstrated by the incorporation of a 10.3 kb insert into the vector. The method reported to accomplish plasmid reconstruction is uniquely versatile yet simple, relying on the strategic placement of primers combined with homologous recombination of PCR products in yeast.

Rapid Dispatches Highlights

BioTechniquesVol. 53, No. 4 Rapid Dispatches HighlightsOpen AccessRapid Dispatches HighlightsNathan BlowNathan BlowSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113933AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit A ‘2 for 1' cloning dealProtein interaction assays are a key approach in understanding protein complexes and cellular signaling. Several such interaction assays, including two-hybrid methods, Forster resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC), have been described and expanded in recent years. One challenge that still exists when using these approaches is obtaining equivalent expression of the potential interacting protein pairs, especially in cases where the fusion proteins are expressed from different plasmids. In an attempt to remedy this issue, Christopher Grefen and Michael Blatt from the University of Glasgow report in Rapid Dispatches a simplified methodology that enable the simultaneous cloning of two genes into two different, independent cloning cassettes. The cloning methodology introduced by the authors takes advantage of site-specific recombination wherein each cassette is flanked by a differing attR site and selection element. To demonstrate the utility of their new cloning approach, the authors constructed a vector for improved ratiometric BiFC (rBiFC) analysis. Here, the two genes of interest are cloned simultaneously into the rBiFC vector, which also possesses a RFP gene under control of the same promoter creating an internal marker for expression control. The authors demonstrate the effectiveness of analyzing protein interactions using their rBiFC system by examining interactions between calcineurin B-like (CBL) proteins and the specific serine-threonine kinases these proteins recruit. Although validated by the authors in a plant system and for ratiomeric BiFC, the cloning approach, as well as the rBiFC assay, should prove applicable to other organisms and systems in the future. See “A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC).”Category: Molecular BiologyStudying gliomas in 3DThe evaluation of cerebral gliomas for malignancy is critical to proper patient survival. Histopathology is mandatory for a definitive diagnosis, but specific immunologic or molecular markers are needed. In a Benchmark article recently published at Rapid Dispatches, a team of researchers led by Lila Kucheryavykh from Universidad Central del Caribe described the use of the fluorescent substrate, 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+), to label glioma cells during the early stages of tumor development. In contrast to the hematoxylin and eosin staining that is often performed when examining tissue slices, ASP+ staining allowed the visualization of 3D structure within living brain slices. In experiments examining the effectiveness of ASP+ staining, glioma cells were implanted into 12-16 week-old mice and staining was performed three weeks post implantation. Imaging of the ASP+ stained tissue allowed reliable 3D visualization of blood vessel configurations and internal tumor cavities for up to two weeks post implantation. The authors did check if ASP+ staining was toxic using a Live/Dead Cytotoxicity kit (it was not), and whether or not the stain also worked with human glioma cells (it did). Taken together, Kucheryavykh et al. demonstrate that ASP+ staining is a simple and effective approach for visualizing gliobastoma tumor cells along with the internal tumor structure and blood vessels, in 3D, in living brain slices during the early stages of tumor development. See “Visualization of implanted GL261 glioma cells in living mouse brain slices using fluorescent 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+).”Category: Microscopy/ImagingCell patterning: where's the tape?When it comes to patterning cells on a surface, researchers often take advantage of microfabrication techniques that are capable of producing specific patterns of cells in the micrometer range. The challenge for general users is that such microfabrication approaches also require a clean room along with photolithography equipment and knowledge of fabrication techniques. While non-photolithography methods have been described, many are limited in scope and still require materials not traditionally found in a lab. In a new Benchmark article in Rapid Dispatches, Raquel Perez-Castillejos and her colleagues at the New Jersey Institute of Technology describe their new approach to patterning mammalian cells using adhesive tape. The authors used standard office tape in combination with poly(dimethylsiloxane) to pattern cells with sub-millimeter precision. In a proof-of-concept experiment, the authors performed a wound-healing assay using their tape approach to place cells. This new method, which is being called tape-based soft lithography, should open the technique of cell patterning to a greater number of labs in the years to come. See “Adhesive-tape soft lithography for patterning mammalian cells: application to wound-healing assays.”Category: Cell BiologyMicrofluidic compartments made by casting PDMS on patterned adhesive tape.(A) Sequence of steps to prepare a master with adhesive tape (steps 1–4) and subsequently, to fabricate a system of microfluidic compartments by replicating the tape-based master with PDMS (steps 5–9).FiguresReferencesRelatedDetails Vol. 53, No. 4 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print October 2012 Information© 2012 Author(s)PDF download

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Deltaku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.

Identification of the Active Site of DS-epimerase 1 and Requirement of N-Glycosylation for Enzyme Function

Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His-205, Tyr-261, and His-450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions, we propose a model where His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction, generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.

Testing transgenic regulatory elements through live mouse imaging

To overcome positional and methylation effects on transgene expression, we developed a universal cloning cassette for in vivo assessment of regulatory elements using the luciferase reporter gene and the CCCD camera. Monitoring luciferase expression pattern in live mice enables screening of large numbers of transgenic founders quickly and inexpensively. We demonstrate that in the engineered transgenic mice, the chicken β-globin 5′HS4 insulator did not always provide the desirable expression pattern, and the Island Element, responsible for the demethylation of the surrounding DNA region, was not beneficial. Both tested liver-specific and developmentally regulated promoters exhibited the expected expression pattern in most transgenic founders.

Targeting the MHC II presentation pathway in allergy vaccine development

The worldwide increase in the incidence of allergic diseases and the limited efficacy of current vaccines require the development of new efficient vaccination strategies. Based on PTD (protein transduction domain) technology, we have engineered MAT (modular antigen translocation) molecules, aimed to enhance antigen presentation through intracellular targeting of the MHC II presentation pathway. MAT vaccines consist of a cloning cassette, which fuses Tat (transactivator of transcription) peptide to a truncated Ii (invariant chain), which is able to target antigens to the nascent MHC II molecules in the trans-Golgi compartment. To test the efficacy of intracellular targeting, we engineered arrays of MAT-fusions and compared the effects of recombinant allergens, Tat-conjugated allergens and MAT-conjugated allergens for the ability to stimulate T-cell proliferation and cytokine production in human PBMC (peripheral blood mononuclear cell) cultures derived from allergic individuals, and to elicit protective immune responses in mice. MAT-vaccines induced a strong proliferation of PBMCs at a low concentration and induced a Th2/Treg (regulatory T-cell) cell shift in the cytokine profile, reflecting those reported in successfully desensitized allergic individuals. In allergic mouse models, we showed that MAT-vaccines are highly efficient in desensitizing mice and protect them from anaphylactic shock. The technology is applicable not only for the treatment of allergies, but also for the development of preventive vaccines in general.

Modular cloning in plant cells

New plant genes are being discovered at a rapid pace. Yet, in most cases, their precise function remains elusive. The recent advent of recombinational cloning techniques has significantly improved our ability to investigate gene functions systematically. For example, proteins fused with diverse fluorescent tags can be expressed at will using versatile cloning cassettes. In addition, novel binary T-DNA vectors are now available to assemble multiple DNA fragments simultaneously, which greatly facilitate plant cell and protein engineering.

High Resolution Crystal Structures of Piscine Transthyretin Reveal Different Binding Modes for Triiodothyronine and Thyroxine

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).

SINGLE-CHAIN Fv ANTIBODY-ALKALINE PHOSPHATASE FUSION PROTEINS PRODUCED BY ONE-STEP CLONING AS RAPID DETECTION TOOLS FOR ELISA

ABSTRACT A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5′ and 3′ end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv–aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv–aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.

A Single Plasmid Vector (pSTAR) Mediating Efficient Tetracycline-Induced Gene Expression

A plasmid vector (pSTAR) has been constructed which confers neomycin resistance for selecting stably transfected cells, possesses a cloning cassette for placing a gene of interest under the control of the tetO DNA motif, and expresses rtTAnls which, upon association with tetracycline, binds to and drives gene expression from the tetO DNA motif. The plasmid pSTAR/LacZ, which has the gene for β-galactosidase inserted into the cloning cassette, was transfected into Chinese hamster ovary (CHO) cells and selected for stably transfected cells. In pooled transfectants of CHO, tetracycline induced the expression of β-galactosidase in 10–30% of cells. Using clonal transfectants, β-galactosidase expression was induced by tetracycline in essentially every cell. Furthermore, induction of β-galactosidase expression by tetracycline was both dose- and time-dependent. Similar tetracycline-induced β-galactosidase expression is also observed in other cell types. The pSTAR vector is thus suited to facilitate the application of tetracycline-induced gene expression in diverse research areas.

An improved GFP cloning cassette designed for prokaryotic transcriptional fusions

A new gfp cloning cassette designed for prokaryotic transcriptional fusions has been constructed. This cassette consists of gfp (containing the S65T `red-shift' [Heim et al. (1995) Nature 373, 663–664] and F64L `protein solubility' [Cormack et al. (1996) Gene 173, 33–38] mutations) flanked by convenient restriction sites, a translational enhancer, and a consensus ribosome binding site with an optimized spacer region. gfp fusion strains containing this cassette demonstrate from 40- to 80-fold greater fluorescence intensity than wild-type gfp fusion strains. Additionally, this cassette confers sufficient fluorescence to recipient cells to be used in low copy-number plasmids, with promoters conferring low levels of transcription, and in bacterial taxa other than Escherichia, such as Pseudomonas.

Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Combining the hok/sok, parDE, and pnd postsegregational killer loci to enhance plasmid stability.

To enhance plasmid segregational stability in bacterial cells, two pairs of independent postsegregational killing loci (genes which induce host killing upon plasmid loss) isolated from plasmids R1, R483, or RP4 (hok+/sok+ pnd+ or hok+/sok+ parDE+) were cloned into a common site of the beta-galactosidase expression vector pMJR1750 (ptac::lacZ+) to form a series of plasmids in which the effect of one or two stability loci on segregational plasmid stability could be discerned. Adding two antisense killer loci (hok+/sok+ pnd+) decreased the specific growth rate by 50% though they were more effective at reducing segregational instability than hok+/sok+ alone. With the ptac promoter induced fully (2.0 mM isopropyl-beta-D-thiogalactopyranoside) and no antibiotic selection pressure, the combination of a proteic killer locus (parDE+) with antisense killer loci (hok+/sok+) had a negligible impact on specific growth rate, maintained high beta-galactosidase expression, and led to a 30 and 190% increase in segregational stability (based on stable generations) as compared to plasmids containing either hok+/sok+ or parDE+ alone, respectively. Use of hok+/sok+ or parDE+ alone with high cloned-gene expression led to ninefold and fourfold increases in the number of stable generations, respectively. Two convenient cloning cassettes have been constructed to facilitate cloning the dual hok+/sok+ parDE+ and hok+/sok+ pnd+ killer systems.