Multicloning Site Research Articles

PUdOs: Concise Plasmids for Bacterial and Mammalian Cells.

The plasmids from the Université d'Ottawa (pUdOs) are 28 small plasmids each comprising one of four origins of replication and one of seven selection markers, which together afford flexible use in Escherichia coli and several related gram-negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators and contains type IIP or IIS restriction sites for conventional or Golden Gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the user's discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors, affording distinct levels of expression and transfection efficiency comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

A recombinant capripoxvirus expressing the F protein of peste des petits ruminants virus and the P12A3C of foot-and-mouth disease virus

BackgroundPeste des petits ruminants (PPR), foot-and-mouth disease (FMD) and sheep pox and goat pox are three important infectious diseases that infect goats, sheep and other small ruminants. It is well-known that the prevention of three diseases rely mainly on their individual vaccines. However, the vaccines have a variety of different disadvantages, such as short duration of immunity, increasing the number of vaccinations, and poor thermal stability. The purpose of this study is to construct a recombinant goat pox virus (rGPV) capable of expressing the F gene of PPRV and the P12A3C gene of FMDV as a live vector vaccine.ResultsThe IRES, FMDV P12A3C and PPRV F genes into the multi-cloning site of the universal transfer plasmid pTKfpgigp to construct a recombinant transfer plasmid pTKfpgigpFiP12A3C, and transfected GPV-infected lamb testis (LT) cells with liposomes and produced by homologous recombination Recombinant GPV (rGPV/PPRVF-FMDVP12A3C, rGPV). The rGPV was screened and purified by green florescence protein (GFP) and xanthine-guanine-phosphoribosyltransferase gene (gpt) of Escherichia coli as selective markers, and the expression of rGPV in LT cells was detected by RT-PCR and immunofluorescence techniques. The results showed that the virus strain rGPV/PPRVF-FMDVP12A3C containing FMDV P12A3C and PPRV F genes was obtained. The exogenous genes FMDV P12A3C and PPRV F contained in rGPV were normally transcribed and translated in LT cells, and the expression products could specifically react with PPRV and FMDV antiserum. Then, the rGPV was intradermally inoculated with goats, the animal experiments showed that rGPV/PPRVF-FMDVP12A3C could induce high levels of specific antibodies against GPV, PPRV and FMDV.ConclusionsThe constructed rGPV induced high levels of specific antibodies against GPV, PPRV and FMDV. The study provides a reference for “ one vaccine with multiple uses “ of GPV live vector vaccine.

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides.

The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. To improve the pMAL expression vector, we introduced the His6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His6 -Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His6 tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His6 and His6 -enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human β-defensin. We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.

New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria.

New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivoassembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium.

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene.

Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.

Genome editing with the donor plasmid equipped with synthetic crRNA-target sequence

CRISPR/Cas-mediated genome editing is a powerful tool for generating genetically mutated cells and organisms. Linearisation of donor cassettes with this system has been shown to facilitate both transgene donor insertion and targeted knock-in. Here, we developed a donor plasmid that we name pCriMGET (plasmid of synthetic CRISPR coded RNA target sequence-equipped donor plasmid-mediated gene targeting), in which an off-target free synthetic CRISPR coded RNA-target sequence (syn-crRNA-TS) is incorporated with a multi-cloning site, where a donor cassette can be inserted. With co-expression of Cas9 and the syn-crRNA-TS guide RNA (gRNA), pCriMGET provides a linearised donor cassette in vivo, thereby promoting the transgene donor insertion and targeted knock-in. When co-injected with Cas9 protein and gRNA into murine zygotes, pCriMGET yielded around 20% transgene insertion in embryos. This method also achieved more than 25% in-frame knock-in at the mouse Tbx3 gene locus without predicted insertion–deletion mutations using a transgene donor with 400-bp homology arms. pCriMGET is therefore useful as a versatile CRISPR/Cas9-cleavable donor plasmid for efficient integration and targeted knock-in of exogenous DNA in mice.

A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts.

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.

Liposome-linked immunosorbent assay enhanced by immuno-PCR using plasmid-encapsulated liposomes

A liposome immunosorbent assay enhanced by immuno-PCR (iPCR-LISA) using antigen-coupled liposomes encapsulating a signal plasmid (pCR-script Amp KS(+)), was developed for the competitive measurement of antigens. The number of signal plasmid DNA molecules per liposome was found to be 2115. Signal plasmid concentration was determined by SYBR-Green real-time PCR, in which the multi-cloning site (MCS) region of the plasmid was amplified using M13 primer sets. The threshold cycle value (CT) was correlated to antigen concentration. A competitive LISA using antigen-coupled liposomes encapsulating a fluorescent marker, carboxyfluorescein (CF), was also performed (CF-LISA). The effects of the measuring conditions and the amount of antigen coupled to liposomes on assay characteristics were determined.The detection limit of the competitive iPCR-LISA was almost the same as that of the competitive CF-LISA. Both the competitive iPCR LISA and CF-LISA could measure a concentration range of 10−7–10−2 μmol/ml, which is much wider than that of 2.7 × 10−5–3.3 × 10−4 μmol/ml using the conventional indirect ELISA method.

Construction of pDUO: A bicistronic shuttle vector series for dual expression of recombinant proteins

Our ability to genetically manipulate microbial systems is often hampered by the availability of genetic tools. Thus, there is a need for the continued expansion of our molecular tool box. In support of this expansion, this study reports the design, construction, and validation of a new bicistronic shuttle vector series, pDUO, for the dual expression of genes in different hosts. Each vector was designed and constructed to contain two araC-pBAD inducible promoter systems for tight control over gene expression. Each araC-pBAD promoter precedes a ribosomal binding site and a multicloning site (MCS). The 5′ end of MCS1 contains a sequence encoding an affinity HIS-tag N-terminus and MCS2 terminates with a sequence encoding an affinity S-tag C-terminus for one-step purification of recombinant proteins encoded by the inserted genes. Both MCS are followed by an rrnBT1 and T2 transcriptional terminator sequence. Each vector in this series also contains a PBR322 and pRO1600-derived replicon to support replication in different host bacteria along with one of four different selectable markers. The functionality of the pDUO vector series was validated through the dual expression of oxalate biosynthetic component (obc) 1 and mrfp in Escherichia coli and Pseudomonas fluorescens. It is anticipated that this new vector series will facilitate functional studies as well as the engineering of bacterial strains for biotechnological purposes.

Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus.

Perkinsus marinus is a protozoan parasite of molluscs that can be propagated in vitro in a defined culture medium, in the absence of host cells. We previously reported that P. marinus trophozoites can be transfected with high efficiency by electroporation using a plasmid based on MOE, a highly expressed gene, and proposed its potential use as a "pseudoparasite." This is a novel gene expression platform for parasites of medical relevance for which the choice of the surrogate organism is based on phylogenetic affinity to the parasite of interest, while taking advantage of the whole engineered surrogate organism as a vaccination adjuvant. Here we improved the original transfection plasmid by incorporating a multicloning site, an enterokinase recognition sequence upstream of GFP, and a His-tag and demonstrate its potential suitability for the heterologous expression of Plasmodium sp. genes relevant to the development of anti-malarial vaccines. Plasmodium berghei HAP2 and MSP8, currently considered candidate genes for a malaria vaccine, were cloned into p[MOE]:GFP, and the constructs were used to transfect P. marinus trophozoites. Within 48 hr of transfection we observed fluorescent cells indicating that the P. berghei genes fused to GFP were expressed. The expression appeared to be transient for both P. berghei genes, as florescence of the transfectants diminished gradually over time. Although this heterologous expression system will require optimization for integration and constitutive expression of Plasmodium genes, our results represent attainment of proof for the "pseudoparasite" concept we previously proposed, as we show that the engineered P. marinus system has the potential to become a surrogate system suitable for expression of Plasmodium spp. genes of interest, which could eventually be used as a malaria vaccine delivery platform. The aim of the present study was to test the ability of marine protozoan parasite P. marinus to express genes of P. berghei .

Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli.

BackgroundThe bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system.ResultsTo fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a “landing pad” (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %.ConclusionsIntegration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Abstract LB-214: DNAJB1-PRKACA chimera increases Aurora kinase A expression in fibrolamellar hepatocellular carcinoma

Abstract Purpose: Treatment of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare liver cancer affecting the pediatric and adolescent population, is limited to surgical resection with no agents or therapeutic modalities available for unresectable disease. There has been recent interest in using Aurora kinase A (AURKA) inhibitors, with a clinical trial evaluating its efficacy in FL-HCC pending. Previously, we described a consistent single deletion of approximately 400 kB in chromosome 19 that results in a chimera between the heat shock protein DNAJB1 and the catalytic subunit of protein kinase A, PRKACA, in all FL-HCC tumor tissue studied. Furthermore, comparative ribonucleic acid sequence analysis has demonstrated that expression of AURKA, a regulator of cytokinesis and mitosis, is increased in FL-HCC tumor tissue compared with adjacent normal tissue. We hypothesize that the DNAJB1-PRKACA chimera is responsible for changes in AURKA expression in FL-HCC. Methods: The DNAJB1-PRKACA chimera was cloned into a lentiviral vector with a fluorescent reporter. Two stable cell lines (HeLa and a hepatocyte cell line, Huh-7) were transduced with a lentivirus containing either an empty multicloning site (control) or the DNAJB1-PRKACA chimera. Transduction was confirmed by the presence of a positive epifluorescent signal and immunoblots showing expression of the chimeric protein. Microarray data were evaluated for changes in AURKA expression while immunoblots were analyzed for the presence of AURKA in all transduced cells. Results: Microarray analysis showed an increase in AURKA transcripts in cells stably expressing the DNAJB1-PRKACA chimera. Compared to cells transduced with the control lentivirus, HeLa and Huh-7 cells transduced with the DNAJB1-PRKACA chimera demonstrated an increase in AURKA on immunoblots. Levels of a loading control, actin, were equivalent, indicating equal levels of protein examined between samples. Conclusion: Expression of the DNAJB1-PRKACA chimera results in an increase in AURKA. Upregulation of AURKA, in turn, may be responsible for subsequent tumor formation in FL-HCC. This finding further supports the DNAJB1-PRKACA chimera's role in the pathogenesis of FL-HCC and provides a potential therapeutic target for a disease that has no effective treatment besides surgery. Citation Format: Irene Isabel P. Lim, Emily A. Greene-Colozzi, Jennifer M. Murphy, Todd E. Heaton, Sanford M. Simon, Michael P. LaQuaglia. DNAJB1-PRKACA chimera increases Aurora kinase A expression in fibrolamellar hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2015-LB-214

Investigating the expression of GFP fused with HIS-TAG at N- or Cterminus using plasmid pHT253 and pHT254 in Bacillus subtilis

pHT253 and pHT254 belong to the plasmid system carrying Pgrac100, a strong promoter, which were commercialized in the global market in 2012 have pushed forward the use of Bacillus subtilis as host for the production of recombinant proteins. Along with strong promoter Pgrac100, these two plasmids were added His-tag to before and after MCS (Multi Cloning Site) in pHT253 (Pgrac100-8xHis-MCS) and pHT254 (Pgrac100- MCS-8xHis), respectively, to facilitate purification steps. Depending on the farget protein, the user selects appropriate effective strategy of fusing His-tag, i.e. either into the N- or the C-temimal of protein. In this study, the GFP gene was used as an indicator gene to investigate the influence of the His-tag position on the protein expression in B. subtilis. Our results showed that the expression of GFP in B. subtilis significantly was reduced in the fusion form with His-tag at the N-terminal. Thiswouldbe important information for the selection of suitable vectors.

An easy way for the rapid purification of recombinant proteins from Helicobacter pylori using a newly designed expression vector.

We constructed a H. pylori expression vector which consisted of both a His-tag and a GST tag as purification tools for recombinant protein and a chloramphenicol resistant cat gene as a reporter. The backbone of the vector pBK contained an ColEI origin of replication and a kanamycin resistant gene. A set of oligos for the His-tag and the PCR product of gst (glutathione S-transferase) gene were inserted sequentially in frame in the multi-cloning site of pBK. The orf of cat was inserted downstream of the gst to generate pBKHGC. The 3' part of H. pylori clpB and flaA were cloned into the vector which was introduced into H. pylori. Recombinant proteins were purified by GSH affinity column, digested with thrombin and were analyzed by western blotting. The final recombinant proteins were successfully purified.

A new tool for cloning and gene expression in Streptococcus pneumoniae

A new replicon suitable for cloning and gene expression was successfully introduced into Streptococcus pneumoniae. The non-integrative lactococcal vectors pIL253 (higher-copy) and pIL252 (lower-copy), which are based on the promiscuous theta-replicating plasmid pAMβ1, were established in pneumococcus. The stability and the small size of these plasmids, together with the presence of a helpful multi-cloning site make them a useful genetic tool for gene expression in this bacterium. The functionality of the system was tested by cloning and expressing the pneumococcal RNase R gene in pIL253. Full constitutive expression of the cloned gene was observed, clearly demonstrating that this plasmid can be used as an expression vector in S. pneumoniae. Moreover, gene expression can be regulated by the use of the lower- or higher-copy number vector versions. The existence of other replicative plasmids based on this family, which are also probably functional in pneumococcus, further broadens the cloning possibilities. We also show that S. pneumoniae cells can accommodate simultaneously pIL252 or pIL253 together with pLS1, a pMV158 derivative, which replicates via a rolling circle mechanism. This fact greatly increases the ability to manipulate this bacterium.The availability of a new family of replicative vectors for genetic manipulation in S. pneumoniae is an important contribution to the study of this pathogenic microorganism.

A simple procedure for protein ubiquitination detection in Saccharomyces cerevisiae: Gap1p as an example

We established a simple procedure for protein ubiquitination detection in Saccharomyces cerevisiae. Enhanced green fluorescent protein (EGFP) was split into two parts, an N-terminal (GN) and a C-terminal (GC) region. The fusion fragments GN-UBI3 and multi-cloning site (MCS)-GC were inserted into the vector pY26-TEF/GPD, resulting in pUbDetec16. pUbDetec16 was designed for use in detecting protein ubiquitination. Any gene of interest can be inserted into the MCS and the recombinant plasmid can be transferred into a Δura3 auxotrophic S. cerevisiae strain. Protein ubiquitination can then be detected using a fluorescence microscope. The ubiquitination of a protein can be determined based on a fluorescence signal. To validate the reliability of this procedure, Gap1p, a protein known to be ubiquitinated, was used as a positive control. A triple mutant of Gap1p, Gap1pK9R,K16R,K76R, which did not contain any ubiquitination site, was used as a negative control. pUbDetec16-GAP1 and pUbDetec16-GAP1K9R,K16R,K76R were constructed and transferred into the Δura3 auxotrophic S. cerevisiae strain CEN.PK2-1D. Transformants of pUbDetec16-GAP1 emitted fluorescence, while the pUbDetec16-GAP1K9R,K16R,K76R transformants did not. The ubiquitination of Gap1p and Gap1pK9R, K16R, K76R was further verified using classical SDS–PAGE analysis. This procedure significantly simplifies manipulation involving ubiquitination detection using the BiFC approach, particularly on a large scale.

Development of a LacZ-based transcriptional reporter system for use with Moraxella catarrhalis

The lack of a transcriptional reporter system for use in Moraxella catarrhalis has hindered studies of gene regulation in this pathogen. PCR and recombinant DNA methods were used to insert a multicloning site (MCS) and promoterless full-length Escherichia coli lacZ gene, flanked by transcriptional terminators both immediately upstream and downstream, into the M. catarrhalis recombinant plasmid pWW115. Insertion into the MCS in the newly constructed plasmid pASE222 of M. catarrhalis promoter regions controlled by either a repressor (i.e., NsrR) or activator (i.e., PhoB) yielded transcriptional fusion constructs that were appropriately responsive to signal inputs dependent on the host strain genotype, as measured quantitatively by means of a Miller β-galactosidase assay. The transcriptional reporter plasmid pASE222 should prove to be a useful tool for rapid screening of factors affecting gene expression in M. catarrhalis.

PLR: A lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters

Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site) – reporter gene system.In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression.

Low copy expression vectors for use in Yersinia sp. and related organisms

In Yersinia, the most commonly used expression vectors for genetic studies such as gene complementation do not effectively allow for both induction and repression of gene expression. Additionally, there is no expression system available that can be induced in bacteria growing in vitro as well as in vivo, e.g. in eukaryotic cell lines or in living animal models. Here, we present a series of novel inducible low copy expression vectors that are well suited for use in the Yersinia species. Their tet operator/promoter/repressor system makes them distinct from other vectors, and gene transcription in bacteria can easily be induced by addition of anhydrotetracyline (ATc) either to the growth medium, to tissue culture medium during bacterial infections of cell lines or by injection into animals infected with bacteria. Researchers can choose between two different antibiotic resistances (kanamycin or spectinomycin), between two copy numbers (5 or 12–22) as well as between two different versions for expression from either the native RBS and ATG or RBS and ATG encoded in the plasmid. The whole vector series contains the same multi-cloning site from pBluescript II KS+ that allows for easy subcloning. Moreover, these vectors are built in a modular fashion that makes it simple to adapt them for other purposes. Finally, in addition to their use in Yersinia they are suitable for use in many other Enterobacteriaceae.