Uninduced Conditions Research Articles

Combining regulated and constitutive protein expression significantly boosts protein expression by increasing productivity without affecting CHO cell growth.

Chinese hamster ovary (CHO) cells are commonly used for the expression of therapeutic proteins. To increase the titer output of CHO production cultures either specific productivity (Qp), growth, or both need to be increased. Generally, Qp and growth are inversely correlated and cell lines with high Qp have slower growth and vice versa. During the cell line development (CLD) process, the faster-growing cells tend to take over the culture and represent the majority of the isolated clones post single cell cloning. In this study, combinations of regulated and constitutive expression systems were used to supertransfect targeted integration (TI) cell lines expressing the same antibody either constitutively or under-regulated expression. Clone screening with a hybrid expression system (inducible + constitutive) allowed identification and selection of higher titer clones under uninduced conditions, without a negative impact on cell growth during clone selection and expansion. Induction of the regulated promoter(s) during the production phase increased the Qp without negatively affecting growth, resulting in approximately twofold higher titers (from 3.5 to 6-7g/L). This was also confirmed using a 2-site TI host where the gene of interest was expressed inducibly from Site 1 and constitutively from Site 2. Our findings suggest that such a hybrid expression CLD system can be used to increase production titers, providing a novel approach for expression of therapeutic proteins with high titer market demands.

Bio-3D printing of scaffold-free osteogenic and chondrogenic constructs using rat adipose-derived stromal cells.

Although autogenous bone implantation is considered to be the gold standard for the reconstruction of bone defects, this approach remains challenging when treating extensive bone defects (EBDs). Therefore, artificial materials (AMs) such as artificial bone and scaffolds are often used for treating EBDs. Nevertheless, complications such as material failure, foreign body reaction, and infection are common. To overcome these issues, we aimed to develop a new treatment for an EBD using scaffold-free adipose-derived stromal cells (ADSCs) to fabricate chondrogenic/osteogenic-induced constructs without AMs. ADSCs were obtained from the subcutaneous adipose tissue of 8-week-old female Wistar rats (n = 3) and assessed to determine their potential for multilineage differentiation into adipocytes (Oil Red O staining), chondrocytes (hematoxylin and eosin, Alcian blue, and Safranin O staining), and osteoblasts (Alizarin red and von Kossa staining). Spheroids (n = 320), each containing 3.0 × 104 ADSCs, were then used to fabricate scaffold-free cell constructs using a bio-3D printer with a needle array. The spheroids and constructs were stimulated with induction medium to induce chondrogenic and osteogenic differentiation. The induced cartilage- and bone-like constructs were finally evaluated using micro-computed tomography (μCT) and histological analysis. The collected ADSCs were capable of trilineage differentiation, and were successfully used to produce scaffold-free constructs. The fabricated constructs (n = 3) exhibited equivalent strength (load, 195.3 ± 6.1 mN; strength, 39.1 ± 1.2 kPa; and stiffness, 0.09 ± 0.01 N/mm) to that of soft tissues such as the muscles in the uninduced condition. In chondrogenic induction experiments, Alcian blue and Safranin O staining confirmed the differentiation of the constructs into cartilage, and cartilage tissue-like structures were produced. In the osteogenic induction experiment, Alizarin Red and von Kossa staining showed calcium salt deposition, and μCT images confirmed the same calcification level as that of the cortical bone. Scaffold-free constructs consisting of ADSCs without an AM were fabricated, and cartilage- and bone-like tissues were successfully generated, demonstrating their potential for bone reconstruction.

Utilization of a tetracycline-inducible system for high-level expression of recombinant proteins in Francisella tularensis LVS

Francisella tularensis is a Gram-negative intracellular pathogen causing tularemia. A number of its potential virulence factors have been identified, but their biology and functions are not precisely known. Understanding the biological and immunological functions of these proteins requires adequate genetic tools for homologous and heterologous expression of cloned genes, maintaining both original structure and post-translational modifications. Here, we report the construction of a new multipurpose shuttle plasmid – pEVbr – which can be used for high-level expression in F. tularensis. The pEVbr plasmid has been constructed by modifying the TetR-regulated expression vector pEDL17 (LoVullo, 2012) that includes (i) a strong F. tularensis bfr promoter, and (ii) two tet operator sequences cloned into the promoter. The cloned green fluorescent protein (GFP), used as a reporter, demonstrated almost undetectable basal expression level under uninduced conditions and a highly dynamic dose-dependent response to the inducer. The utility of the system was further confirmed by cloning the gapA and FTT_1676 genes into the pEVbr vector and quantifying proteins expression in F. tularensis LVS, as well as by studying post-translational modification of the cloned genes. This study demonstrates that high levels of recombinant native-like Francisella proteins can be produced in Francisella cells. Hence, this system may be beneficial for the analysis of protein function and the development of new treatments and vaccines.

A low-background Tet-On system based on post-transcriptional regulation using Csy4.

On account of its stringent regulation and high rate of induction, the tetracycline regulatory system is used extensively for inducing target gene expression in eukaryotes. However, under certain circumstances, its associated background expression can be problematic, as in the expression of highly toxic proteins. We found that when using the Tet-On 3G system to drive expression of the kid toxin gene in sf9 insect cells, a higher percentage of cells were killed than when using an empty vector in the absence of the induction agent doxycycline, thereby indicating the leaky expression of this inducible expression system. Moreover, we found that the tetracycline-controlled transcriptional silencer (tTS) does not effectively reduce the background expression of the Tet-On 3G system in sf9 cells. However, Csy4, a Cas9 homologous protein in the CRISPR family with sequence-specific endonuclease activity, was found to be effective in reducing the Tet-On 3G system-associated background expression, although there was a concomitant reduction in the maximum induced expression. Nevertheless, we found that modification of the system via incorporation of TRE-controlled anti-sense csy4 in combination with a WSSVie1 (Δ23) promotor-driven sense csy4 significantly reduced the leaky expression of the Tet-On 3G system, and that the level of induction was higher than that initially obtained. This optimized Tet-On 3G system can significantly reduce cell death attributed to the background expression of Kid under uninduced conditions. Therefore, we developed a novel low-background inducible expression system for use in insect cells and potentially in other organisms including mammals based on post-transcriptional regulation using Csy4.

Interaction of LysM BON family protein domain with carbapenems: A putative mechanism of carbapenem resistance

Carbapenem resistance in Gram-negative pathogens has become a global concern for health workers worldwide. In one of our earlier studies, a Klebsiella pneumoniae-carbapenemase-2 producing strain was induced with meropenem to explore differentially expressed proteins under induced and uninduced conditions. There is, LysM domain BON family protein, was found over 12-fold expressed under the induced state. A hypothesis was proposed that LysM domain protein might have an affinity towards carbapenem antibiotics making them unavailable to bind with their target. Hence, we initiated a study to understand the binding mode of carbapenem with LysM domain protein. MICs of imipenem and meropenem against LysM clone were increased by several folds as compared to NP-6 clinical strain as well as DH5 α (PET-28a KPC-2) clone. This study further revealed a strong binding of both antibiotics to LysM domain protein. Molecular simulation studies of LysM domain protein with meropenem and imipenem for 80 ns has also showed stable structure. We concluded that overexpressed LysM domain under induced condition interacted with carbapenems, leading to enhanced resistance as proved by high MIC values. Hence, the study proved the proposed hypothesis that the LysM domain plays a significant role in the putative mechanism of antibiotics resistance.

Evaluation of the Effect of Promoter Type on the Immunogenicity of the Live Recombinant Salmonella Vaccines Expressing Escherichia Coli Heat-labile Enterotoxins (LTB).

Enterotoxigenic Escherichia coli (ETEC)-induced diarrhoea is the second most common cause of death in children in the developing countries. Heat labile toxin (LT) is responsible for ETEC-induced diarrhoea. In the present study, a novel live ETEC vaccine based on subunit B of LT (LTB) expression in attenuated PhoPc Salmonella strain was developed. Herein, we aimed to compare the in-vitro activity of promoters including constitutive tac, IPTG inducible trc, and in-vivo-inducible (nirB and nirB78-23) in PhoPc. Additionally, the ability of these recombinant PhoPc/pLTBs to induce LTB-specific antibody responses in BALB/c mice after nasal immunization was evaluated. In-vitro studies demonstrated that PhoPc has the ability to produce rLTB. Furthermore, nirB promoter directed significantly more LTB expression in PhoPc/pnirBLTB under anaerobic condition without induction compared to the amount of rLTB secreted by PhoPc/ptrcLTB in bacterial soup under uninduced condition (6.06 ± 0.05 vs. 1.4 ± 0.46 μg/109 cfu, p < 0.01). In addition, the constitutive rLTB expression from tac promoter was more than its expression from uninduced trc promoter in bacterial soup (4.2 ± 0.92 vs. 1.4 ± 0.46 (μg/109 cfu)) and pellet (27.4 ± 0.89 vs. 13.4 ± 1.42 (μg/109 cfu), p < 0.0001). However, the mice immunized with PhoPc/ptrcLTB elicited the superior anti-LTB responses among the PhoPc containing the examined prompters, which were significantly higher than those induced by PhoPc/pnirB78-23LTB and PhoPc/pnirB, 6 weeks after the first immunization. Totally, it could be concluded that in-vitro analysis of promoters for LTB expression in PhoPc may not necessarily predict the recombinant PhoPc immunogenicity.

Pseudomonas aeruginosa nfuA: Gene regulation and its physiological roles in sustaining growth under stress and anaerobic conditions and maintaining bacterial virulence.

The role of the nfuA gene encoding an iron-sulfur ([Fe-S]) cluster-delivery protein in the pathogenic bacterium Pseudomonas aeruginosa was investigated. The analysis of nfuA expression under various stress conditions showed that superoxide generators, a thiol-depleting agent and CuCl2 highly induced nfuA expression. The expression of nfuA was regulated by a global [2Fe-2S] cluster containing the transcription regulator IscR. Increased expression of nfuA in the ΔiscR mutant under uninduced conditions suggests that IscR acts as a transcriptional repressor. In vitro experiments revealed that IscR directly bound to a sequence homologous to the Escherichia coli Type-I IscR-binding motifs on a putative nfuA promoter that overlapped the -35 element. Binding of IscR prevented RNA polymerase from binding to the nfuA promoter, leading to repression of the nfuA transcription. Physiologically, deletion of nfuA reduced the bacterial ability to cope with oxidative stress, iron deprivation conditions and attenuated virulence in the Caenorhabditis elegans infection model. Site-directed mutagenesis analysis revealed that the conserved CXXC motif of the Nfu-type scaffold protein domain at the N-terminus was required for the NfuA functions in conferring the stress resistance phenotype. Furthermore, anaerobic growth of the ΔnfuA mutant in the presence of nitrate was drastically retarded. This phenotype was associated with a reduction in the [Fe-S] cluster containing nitrate reductase enzyme activity. However, NfuA was not required for the maturation of [Fe-S]-containing proteins such as aconitase, succinate dehydrogenase, SoxR and IscR. Taken together, our results indicate that NfuA functions in [Fe-S] cluster delivery to selected target proteins that link to many physiological processes such as anaerobic growth, bacterial virulence and stress responses in P. aeruginosa.

Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering

Plasmid-based systems are the most appropriate for multistep lambda Red (λRed)-mediated recombineering, such as the assembly of strains for biotechnological applications. Currently, the widely used λRed-expressing plasmids use a temperature-sensitive origin of replication or temperature shift control of λRed expression. In this work, we have constructed a new, conditionally replicating vector that can be efficiently eliminated from the host strain through passaging in medium containing isopropyl-β-d-thiogalactopyranoside. Using the new vector, we have developed two improved helper plasmids (viz., pDL17 and pDL14) for dsDNA and oligonucleotide-mediated recombineering, respectively. The plasmid pDL14 contains a dominant negative mutSK622A allele that suppresses methyl-directed mismatch repair (MMR). The coexpression of λRed and mutSK622A provides efficient oligonucleotide-mediated recombineering in the presence of active host MMR. The expression of λRed was placed under the control of the tightly regulated PrhaB promoter. Because of their low expression level under uninduced conditions, both plasmids could be maintained without elimination for multiple recombineering steps. The temperature-independent replication of the plasmids and control of λRed expression by l-rhamnose allow for all procedures to be performed at 37 °C. Thus, the new plasmids are robust, convenient, and versatile tools for Escherichia coli genome editing.

New Shuttle Vectors for Gene Cloning and Expression in Multidrug-Resistant Acinetobacter Species.

Understanding bacterial pathogenesis requires adequate genetic tools to assess the role of individual virulence determinants by mutagenesis and complementation assays, as well as for homologous and heterologous expression of cloned genes. Our knowledge of Acinetobacter baumannii pathogenesis has so far been limited by the scarcity of genetic tools to manipulate multidrug-resistant (MDR) epidemic strains, which are responsible for most infections. Here, we report on the construction of new multipurpose shuttle plasmids, namely, pVRL1 and pVRL2, which can efficiently replicate in Acinetobacter spp. and in Escherichia coli The pVRL1 plasmid has been constructed by combining (i) the cryptic plasmid pWH1277 from Acinetobacter calcoaceticus, which provides an origin of replication for Acinetobacter spp.; (ii) a ColE1-like origin of replication; (iii) the gentamicin or zeocin resistance cassette for antibiotic selection; and (iv) a multilinker containing several unique restriction sites. Modification of pVRL1 led to the generation of the pVRL2 plasmid, which allows arabinose-inducible gene transcription with an undetectable basal expression level of cloned genes under uninduced conditions and a high dynamic range of responsiveness to the inducer. Both pVRL1 and pVRL2 can easily be selected in MDR A. baumannii, have a narrow host range and a high copy number, are stably maintained in Acinetobacter spp., and appear to be compatible with indigenous plasmids carried by epidemic strains. Plasmid maintenance is guaranteed by the presence of a toxin-antitoxin system, providing more insights into the mechanism of plasmid stability in Acinetobacter spp.

Global Expression Analysis of Quorum Sensing-Controlled Genes by RNAseq.

RNA sequencing (RNAseq) enables transcriptional profiling of many organisms. This chapter describes the use of RNAseq in prokaryotes to identify quorum sensing (QS)-controlled transcripts by comparing samples from QS-induced and -uninduced conditions. Briefly, each RNA sample is converted to ds-cDNA in a method that limits amplification of ribosomal RNA species. The ds-cDNA contains adapters that enable sequencing and quantification by next-generation sequencing (NGS).

Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae.

Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes.

Evolutionary Design of Choline-Inducible and -Repressible T7-Based Induction Systems.

By assembly and evolutionary engineering of T7-phage-based transcriptional switches made from endogenous components of the bet operon on the Escherichia coli chromosome, genetic switches inducible by choline, a safe and inexpensive compound, were constructed. The functional plasticity of the BetI repressor was revealed by rapid and high-frequency identification of functional variants with various properties, including those with high stringency, high maximum expression level, and reversed phenotypes, from a pool of BetI mutants. The plasmid expression of BetI mutants resulted in the choline-inducible (Bet-ON) or choline-repressible (Bet-OFF) switching of genes under the pT7/betO sequence at unprecedentedly high levels, while keeping the minimal leaky expression in uninduced conditions.

The transcriptional activator Ino2p dissociates from the yeast INM1 promoter in induction.

Mood stabilizers lithium and valproates are widely used in the treatment of bipolar disorder. It has been shown that these drugs can affect the inositol monophosphatase activity and thus the inositol de novo biosynthesis. However, the molecular mechanism of this action has thus far been vague. As such, characterizing the regulation of the gene encoding inositol monophosphatase at the molecular level can help to understand the bipolar disorder. As the model organism, the inositol monophosphatase is encoded by INM1 in Saccharomyces cerevisiae. In this study, we showed, using real-time reverse transcriptase polymerase chain reaction analysis, that INM1 is expressed in the presence of inositol, suggesting that the presence of inositol is required for INM1 transcriptional activation. We also demonstrated, using chromatin immunoprecipitation, that Ino2p is present at the promoter under uninduced conditions. Upon induction, Ino2p dissociates from the INM1 promoter. Furthermore, chromatin remodelers Ino80p and Snf2p are recruited to INM1 promoter upon induction as well as histone acetylases Gcn5p and Esa1p. Altogether, we have provided the evidence which describes how the transcriptional activator and coactivators participate in INM1 activation.

Interplay of transcriptional and small RNA‐dependent control mechanisms regulates chitosugar uptake in Escherichia coli and Salmonella

Escherichia coli and Salmonella can use chitin-derived oligosaccharides as carbon and nitrogen sources. Chitosugars traverse the outer membrane through a dedicated chitoporin, ChiP, and are transported across the cytoplasmic membrane by the chitobiose transporter (ChbBCA). Previous work revealed that synthesis of the chitoporin, ChiP, requires transcription of the chbBCARFG operon. A sequence from the chbBC portion of the transcript was shown to act as a decoy target for a regulatory small RNA, ChiX, that normally blocks chiP expression. ChiX is destabilized and degraded upon pairing with chbBC RNA. Here, we show that the chiP gene, like the chbBCARFG operon, is also downregulated at the transcriptional level by the NagC repressor. NagC repression is critical in maintaining chiP mRNA levels low enough, relative to ChiX, to allow full silencing by this sRNA. We also show that pairing of ChiX to chbBC RNA downregulates chbC under uninduced conditions, that is, when ChiX is in excess to the decoy sequence. Hence, under these conditions, chbBC RNA is not just a decoy, but a true target of ChiX regulation. Altogether these findings underscore the importance of stoichiometry in dictating the strength of the sRNA response and in differentiating the regulator from the regulatory target.

Comprehensive Cysteine-scanning Mutagenesis Reveals Claudin-2 Pore-lining Residues with Different Intrapore Locations

The first extracellular loop (ECL1) of claudins forms paracellular pores in the tight junction that determine ion permselectivity. We aimed to map the pore-lining residues of claudin-2 by comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45 cysteine mutations within the ECL1 by expression in polyclonal Madin-Darby canine kidney II Tet-Off cells and found nine mutants that displayed a significant decrease of conductance after treatment with the thiol-reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate, indicating the location of candidate pore-lining residues. Next, we stably expressed these candidates in monoclonal Madin-Darby canine kidney I Tet-Off cells and exposed them to thiol-reactive reagents. The maximum degree of inhibition of conductance, size selectivity of degree of inhibition, and size dependence of the kinetics of reaction were used to deduce the location of residues within the pore. Our data support the following sequence of pore-lining residues located from the narrowest to the widest part of the pore: Ser(68), Ser(47), Thr(62)/Ile(66), Thr(56), Thr(32)/Gly(45), and Met(52). The paracellular pore appears to primarily be lined by polar side chains, as expected for a predominantly aqueous environment. Furthermore, our results strongly suggest the existence of a continuous sequence of residues in the ECL1 centered around Asp(65)-Ser(68) that form a major part of the lining of the pore.

Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pmpromoter

BackgroundXylS is the positive regulator of the inducible Pm promoter, originating from Pseudomonas putida, where the system controls a biochemical pathway involved in degradation of aromatic hydrocarbons, which also act as inducers. The XylS/Pm positive regulator/promoter system is used for recombinant gene expression and the output from Pm is known to be sensitive to the intracellular XylS concentration.ResultsBy constructing a synthetic operon consisting of xylS and luc, the gene encoding luciferase, relative XylS expression levels could be monitored indirectly at physiological concentrations. Expression of XylS from inducible promoters allowed control over a more than 800-fold range, however, the corresponding output from Pm covered only an about five-fold range. The maximum output from Pm could not be increased by introducing more copies of the promoter in the cells. Interestingly, a previously reported XylS variant (StEP-13), known to strongly stimulate expression from Pm, caused the same maximum activity from Pm as wild-type XylS at high XylS expression levels. Under uninduced conditions expression from Pm also increased as a function of XylS expression levels, and at very high concentrations the maximum activity from Pm was the same as in the presence of inducer.ConclusionAccording to our proposed model, which is in agreement with current knowledge, the regulator, XylS, can exist in three states: monomers, dimers, and aggregates. Only the dimers are active and able to induce expression from Pm. Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates. Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression. The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

Strain Improvement by Overexpression of the laeA Gene in Monascus pilosus for the Production of Monascus-Fermented Rice

Monascus species have been used to produce fermented rice called Monascus-fermented rice (MFR). To improve a Monascus strain via activation of secondary metabolite (SM) gene clusters for use in the production of MFR, we overexpressed an ortholog of the laeA gene, which encodes a global positive regulator of secondary metabolism under the control of the strong heterologous Aspergillus nidulans alcA promoter in Monascus pilosus. The OE::laeA transformant produced more SMs, including those not detected under uninduced conditions. MFR produced using the M. pilosus OE::laeA strain contained 4 times more monacolin K, a cholesterol-lowering agent, than MFR produced using the wild-type strain. In addition, pigment production was remarkably increased, and the antioxidant activity was increased as well. The results from this study suggest that Monascus species, which are important industrial fermentative fungi in Asia, can be improved for the production of functional foods by overexpressing the laeA gene.

Construction of a tightly-controlled expression system for use in Pseudomonas

Transcriptional analysis of czcCBA gene cluster in Pseudomonas aeruginosa PAO1 revealed that the promoter of this operon is tightly controlled by Zn. The promoter activity is undetectable in the uninduced condition but reaches high levels when induced. We used the czcCBA promoter to construct a tightly-controlled expression vector series, pLY vectors (pLY-A, pLY-B and pLY-C). These differed by just one base pair at the multiple cloning sites, allowing convenient in-frame cloning of any genes for expression. Using the luxCDABE reporter, this expression system was shown to be controlled by Zn in a dose-dependent manner in Pseudomonas; cloning the sacB gene encoding levansucrase onto the pLY-C vector enabled Zn-controlled growth of P. aeruginosa PAO1. These pLY vectors provide a useful tool for functional characterization of genes and controlled production of difficult or toxic proteins in Pseudomonas sp.

AACA Element Negatively Regulates Expression of Protein Phosphatase 2C (&amp;lt;i&amp;gt;PP2C&amp;lt;/i&amp;gt;) like Promoter in &amp;lt;i&amp;gt;Arabidopsis thaliana&amp;lt;/i&amp;gt;

Genome wide analysis of Arabidopsis thaliana reveals a unique genetic arrangement of ACGT motif in the protein phosphatase 2C (PP2C) like promoter (accession number AT5G59220). In the present study, full length, 900 base pair, 500 base pair, 400 base pair and NRM deletion variants of PP2C like promoter were constructed to investigate the activity of PP2C like promoter in the presence of inducers like abscisic acid, jasmonic acid and salicylic acid. The PP2C promoter has three ACGT elements in close vicinity. They are so positioned that they are separated by a spacer of 30 base pair and 5 base pair respectively from each other. The study has shown that ACGTN30ACGT genetic architecture is essential for the promoter to be induced in response to abscisic acid. The synergistic and antagonistic effects of cis elements were observed. AACA is a positive regulatory element in endosperm and is known to act as negative regulatory element in other tissues. In this study AACA, have been found to negatively regulate the expression of reporter gene EGFP in both induced and under uninduced conditions.