Blue Colonies Research Articles

A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products

Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.

Spectra MRSA, a New Chromogenic Agar Medium To Screen for Methicillin-ResistantStaphylococcus aureus

A novel chromogenic medium, Spectra MRSA (Remel, Lenexa, KS), was designed to detect methicillin-resistant Staphylococcus aureus (MRSA) rapidly and more efficiently than traditional media (i.e., tryptic soy agar with 5% sheep blood [SBA] and mannitol salt agar [MSA]). A multicenter study (including four clinical trial sites and the Medical College of Wisconsin [MCW] Milwaukee, WI) compared the performance characteristics of Spectra MRSA to those of the traditional media for the detection of MRSA. For this study, 767 nasal swab specimens from the multicenter study (traditional medium used, SBA) and 667 nasal swab specimens from MCW (traditional medium used, MSA) were plated on each test medium and examined after 24 and 48 h of incubation. At 24 h, the sensitivity and the specificity of each medium were as follows: in the multicenter study, 95.4% and 99.7%, respectively, for Spectra MRSA and 93.6% and 100%, respectively, for SBA; at MCW, 95.2% and 99.5%, respectively, for Spectra MRSA and 88.7% and 94.0%, respectively, for MSA. The positive predictive values of each medium at 24 h were as follows: in the multicenter study, 98.1% for Spectra MRSA and 100% for SBA; at MCW, 95.2% for Spectra MRSA and 60.4% for MSA. In our evaluation, we found that Spectra MRSA was able to rapidly identify and differentiate methicillin-resistant S. aureus from methicillin-susceptible S. aureus on the basis of the utilization of chromogens that result in denim blue colonies, thus eliminating the need for biochemical analysis and antimicrobial susceptibility testing. Extending the incubation beyond 24 h did not significantly improve the recovery of MRSA and resulted in decreased specificity.

Effect of reaction conditions and 3AB on the mutation rate of poliovirus RNA-dependent RNA polymerase in a α-complementation assay

The fidelity of poliovirus RNA-dependent RNA polymerase (3Dpol) was determined using a modified α-complementation assay. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. Various conditions including high and low MgCl 2, replacing MgCl 2 with MnCl 2, skewed nucleotide pools, and the presence of poliovirus protein 3AB were analyzed. The assay included RNA synthesis by 3Dpol on an RNA template that coded for a region of the alpha peptide of β-galactosidase (lacZ-α). The product of this reaction was used as a template for a second round of 3Dpol synthesis and the resulting RNA was reverse transcribed to DNA by reverse transcriptase. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for β-galactosidase activity by blue–white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on lacZ-α. Although 3AB strongly stimulated 3Dpol synthesis as expected, no change in fidelity was detected. Changes in MgCl 2 also showed little effect. Mutation rates of ∼9 × 10 −5 (∼1 error per 11,000 incorporations) were estimated for these conditions. In contrast, MnCl 2 or skewed nucleotide pools were highly mutagenic resulting in lowered fidelity.

An advanced blue–white screening method for construction of shRNA expression vectors

Short hairpin RNA (shRNA) encoded within an expression vector is an effective tool for exploration of gene function in mammalian cells. Many of the current methods for constructing shRNA expression vectors require cumbersome and time-consuming procedures for identification of the desired recombinants. We have developed a highly efficient and less labor-intensive cloning method that allows the construction of shRNA expression vectors in one day and with minimal effort. This advanced blue–white screening technique was developed by combining the reconstitution of ideal lacO with TA cloning. The DNAs are simply ligated into the destination vectors and, following transformation, a desired recombinant event will give a typical blue colony. In addition, we have used this cloning method for the construction of targeting reporter expression vectors to measure the efficacy of the corresponding shRNA. We constructed 122 functional shRNA expression vectors and sequencing of the positive cloning vectors confirmed a high degree of accuracy. Only three short DNA primers are needed for constructing both shRNA and targeting reporter expression vectors. This advanced blue–white screening system is a powerful tool for the high-throughput assay of RNAi libraries.

An Efficient Method to Prepare PCR Cloning Vectors

An improved procedure for preparing PCR cloning vectors was developed. This procedure includes the incorporation of adapters to create XcmI restriction enzyme sites in pBluescript II SK(+) vectors, digestion with XcmI followed by further digestion of the small fragment produced by XcmI digestion with additional enzymes, and purification with PCR purification kits. Using this procedure, PCR cloning vectors with high ligation efficiencies and low blue or false-positive colonies were obtained.

Microbicidal Action of Indole-3-Acetic Acid Combined with Horseradish Peroxidase on Prototheca zopfii from Bovine Mastitis

We investigated the toxic effect of indole-3-acetic acid (IAA) combined with horseradish peroxidase (HRP) on Prototheca zopfii from bovine mastitis. P. zopfii isolates were identified and characterized by morpho-physiological parameters; presences of P. zopfii genotype 2 were also investigated. Subsequently, P. zopfii was incubated in the absence (control) or presence of IAA/HRP and examined for: (i) cell viability; (ii) colonies number formation; (iii) antioxidant enzyme activity; and (iv) DNA integrity. Significance of differences was calculated using ANOVA and Tukey's test (P < or = 0.05). As evidenced by Trypan blue exclusion and colony formation in Sabouraud dextrose agar, IAA/HRP addition to the culture reduced respective P. zopfii viability and P. zopfii colony formation in a concentration- and time-dependent manner. IAA/HRP specifically reduced cell viability in 10, 15, 20, 25, and 32% after 4, 6, 8, 10, and 12 h of incubation, respectively, compared with the control at the same time. The number of colony formation was inhibited (45, 82, and 88%) by IAA/HRP after 4, 6, and 9 h of incubation, respectively, compared with the control at the same time. In addition, P. zopfii antioxidant activity increased measurably in the presence of IAA/HRP (6 h); superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase increased by 90, 120, 150% and 3.4 times, compared with the controls. IAA/HRP did not appear to effect P. zopfii DNA integrity when examined by electrophoresis. In conclusion, IAA/HRP appears to function as a microbicidal mechanism on P. zopfii genotype 2 from bovine mastitis.

Localisation of DivIVA by targeting to negatively curved membranes

DivIVA is a conserved protein in Gram-positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

Performance of CHROM Agar and Oxacillin Resistant Screening Agar Base Media for Detection of Methicillin Resistant Staphylococcus aureus (MRSA) from Chronic Wound

CHROM agar Staphylococcus aureus and Oxacillin Resistant Screening Agar Base (ORSAB) media with oxacillin were evaluated for the screening of Methicillin Resistant Saphylococcus aureus (MRSA). Among 190 samples, totally 126 confirmed Staphylococcus aureus strains were used for screening of MRSA used CHROM agar and ORSAB media were compared with the other MRSA screening media like Baird Park agar (BPA) with ciprofloxacin, Mannitol salt agar (MSA) with oxacillin, Blood agar (BA) with oxacillin and Muller Hinton agar (MHA) with oxacillin. Totally 54 MRSA strains were confirmed using PCR, among that 83% and 92% of the MRSA stains were isolated as mauve colonies on CHROM agar and blue colonies on ORSAB medium in 24 hrs incubation, compare with 64%, 61%, 50%, and 42% of the strains that were isolated on BPA, MSA, BA and MHA respectively. After 48 hrs of incubation 100%, 98%, 77%, 77%, 72% and 68% of the MRSA strains were isolated on CHROM agar, ORSAB, BPA, MSA, BA and MHA respectively. CHROM agar Staphylococcus aureus and ORSAB agar proved to be more sensitivity and specificity than other MRSA selective media. These provide an alternative for the detection of MRSA in clinical laboratories, especially when PCR is unavailable.

Detection of bacterioplankton in immersed cadavers using selective agar plates

We measured bacterioplankton in blood from cadavers retrieved from the sea ( n = 12), near estuaries ( n = 4), rivers (fresh water, n = 8) and from bathtubs ( n = 4) as well as from non-drowned victims ( n = 10) discovered near aquatic environments. Blood from 11 victims drowned in seawater developed bioluminescent and/or blue colonies (oxidase test positive) on selective media containing 2–4% NaCl. Homology analyses of the 16S rRNA gene showed that all of them were marine bacteria (genera: Photobacterium, Vibrio, Shewanella, Psychrobacter). Blood from all victims drowned in rivers generated blue colonies on plates containing ⩽3%, but not 4% NaCl. Homology analyses showed that the blue colonies were generated from bacteria that inhabit fresh water ( Aeromonas). None of the blood samples from victims that drowned in bathtubs generated bioluminescent and blue colonies. However, all cadavers contained bacteria that produced unstained colonies ( Staphylococcus, Bacillus, Enterobacter, Escherichia, etc.). Among non-drowned victims, blood from two gave rise to blue colonies on plates containing ⩽3% NaCl ( Pseudomonas). Of the cadavers found near estuaries, bioluminescent and blue colonies developed from two of them on media containing 2–4% NaCl ( Photobacterium, Vibrio, Listonella), but not from two others on plates containing 4% NaCl (at ⩽3%; blue colonies, Aeromonas; unstained colonies, Citrobacter, Vagococcus, Proteus, Enterobacter). These results suggested that the presence of numerous bacterioplankton in immersed cadavers could support a conclusion of death by drowning.

A novel way to purify recombinant baculoviruses by using bacmid

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.

Simple and rapid detection of histamine-forming bacteria by differential agar medium

The simple and rapid detection of histamine-forming bacteria (HFB) was achieved using a newly developed filtration method. In this method not only liquid samples including seawater, but also seafood, can be assayed on-site. Liquid samples were filtered through a two-layer filtration system with an upper 10-μm pore size membrane filter ( φ 25 mm) and lower 0.2-μm pore size, spherical membrane filter ( φ 25 mm). In the case of seafood, samples were minced with nine volumes (v/w) of sterilized saline solution in aseptic conditions, and the homogenates and their dilutions were filtered through the two-layer system. The two-layer membrane filter was then rinsed with 10 ml sterilized saline solution. The lower membrane filter was placed on an artificial seawater agar medium plate (pH 5.8), containing histidine (0.5%) as a carbon source and bromothymol blue (0.04%) as a pH indicator, and incubated at 35 °C. HFB were detected as blue halo colonies on the lower membrane filter after 5 h of incubation. The color change in agar medium is attributed to the alkaline shift following histamine formation by HFB. Histamine-producing ability for colonies with a blue halo was confirmed by prolonged incubation in histidine broth medium at 35 °C for 24 h. This simple filtration method can rapidly detect HFB and can be applied to food hygiene systems, including the hazard analysis and control critical point (HACCP) system in seafood processing lines.

Vitamin C Antagonizes the Cytotoxic Effects of Antineoplastic Drugs

Vitamin C is an antioxidant vitamin that has been hypothesized to antagonize the effects of reactive oxygen species-generating antineoplastic drugs. The therapeutic efficacy of the widely used antineoplastic drugs doxorubicin, cisplatin, vincristine, methotrexate, and imatinib were compared in leukemia (K562) and lymphoma (RL) cell lines with and without pretreatment with dehydroascorbic acid, the commonly transported form of vitamin C. The effect of vitamin C on viability, clonogenicity, apoptosis, P-glycoprotein, reactive oxygen species (ROS), and mitochondrial membrane potential was determined. Pretreatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by trypan blue exclusion and colony formation after treatment with all antineoplastic agents tested. Vitamin C given before doxorubicin treatment led to a substantial reduction of therapeutic efficacy in mice with RL cell-derived xenogeneic tumors. Vitamin C treatment led to a dose-dependent decrease in apoptosis in cells treated with the antineoplastic agents that was not due to up-regulation of P-glycoprotein or vitamin C retention modulated by antineoplastics. Vitamin C had only modest effects on intracellular ROS and a more general cytoprotective profile than N-acetylcysteine, suggesting a mechanism of action that is not mediated by ROS. All antineoplastic agents tested caused mitochondrial membrane depolarization that was inhibited by vitamin C. These findings indicate that vitamin C given before mechanistically dissimilar antineoplastic agents antagonizes therapeutic efficacy in a model of human hematopoietic cancers by preserving mitochondrial membrane potential. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.

Screening for proteins interacting with MCM7 in human lung cancer library using yeast two hybrid system.

MCM7 is a subunit of the MCM complex that plays a key role in DNA replication initiation. But little is known about its interaction proteins. In this study yeast two hybrid screening was used to identify the MCM7 interacting proteins. Yeast expression vector containing human full length MCM7-pGBKT7 plasmid was constructed, and with a library of cDNAs from human lung cancer-pACT2 plasmid was transformed into yeast strain AH109, and was electively grew in X-a-gal auxotrophy medium SD/-Trp-Leu-His-Ade, and the blue colonies were picked up, the plasmid of the yeast colonies was extracted , and transformed into E. Coli to extract DNA and performed sequence analysis. Eleven proteins were identified which could specifically interact with MCM7 proteins, among these five were cytoskeleton proteins, six were enzymes, kinases and related receptors. The investigation provides functional clues for further exploration of MCM7 gene.

Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.

An evaluation of fluorogenic and chromogenic assays for the direct enumeration of Escherichia coli

Three direct plating methods to enumerate Escherichia coli from food in 24 h are described. Unlike the majority of enterics, 96% of E. coli are able to cleave β‐glucuronic acid. This reaction can be observed by incorporating (1) 4‐methylumbelliferyl‐β‐D‐glucuronate, (2) para‐nitrophenyl‐β‐D‐glucuronate or (3) 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronate (BCIG) into a peptone tergitol agar base. Escherichia coli produce fluorescent, yellow or deep blue colonies from these three compounds respectively. BCIG agar proved the easiest to read and produced the least number of false positives and false negatives. An attempt is made to explain the poor correlation achieved between this method and a conventional most probable number technique.

Marine bacteria comprise a possible indicator of drowning in seawater

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples ( n = 20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater ( n = 8) and in fresh water ( n = 7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n = 7). Bioluminescent colonies were detectable on 2–4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria ( Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.

Lodderomyces elongisporus Masquerading as Candida parapsilosis as a Cause of Bloodstream Infections

Ten yeast bloodstream isolates identified as Candida parapsilosis by conventional methods grew as turquoise blue colonies on Chromagar media. Subsequent sequence analysis showed that these isolates were the species Lodderomyces elongisporus. To our knowledge, this is the first published report of L. elongisporus as a cause of human disease.

A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium

The mechanisms that promote and regulate transcription in mycoplasmas are poorly understood. Here, a promoter-probe vector based on the pMTnTetM438 minitransposon and containing a promoterless lacZ reporter gene was constructed to analyse Mycoplasma genitalium transcription in vivo. Recovered transposon insertions were in monocopy, with 16 % expressing enough beta-galactosidase (beta-Gal) to yield colonies exhibiting a detectable blue colour. A sample of 52 blue colonies was propagated and selected for further analyses. The beta-Gal activity of the corresponding cultures was measured to quantify, in a reproducible way, the transcription levels of the interrupted ORFs. Several insertions were found in sense with the interrupted ORF, but surprisingly there was also a number of insertions in non-coding regions, many of them in repetitive DNA regions known as MgPa islands. Moreover, 30 % of the analysed transposon insertions had the lacZ gene in the opposite orientation to the coding frame, suggesting the existence of antisense transcripts that may be involved in the control of gene expression in M. genitalium.

A Programmable Biomolecular Computing Machine with Bacterial Phenotype Output

The main advantage of autonomous biomolecular computing devices over electronic computers is their ability to interact directly with biological systems. No interface is required since all components of molecular computers, including hardware, software, input, and output are molecules that interact in solution along a cascade of programmable chemical events. Here, we demonstrate for the first time that the output of a computation preduced by a molecular finite automaton can be a visible bacterial phenotype. Our 2-symbol-2-state finite automaton utilized linear double-stranded DNA inputs that were prepared by inserting a string of six base pair symbols into the lacZ gene on the pUC18 plasmid. The computation resulted in a circular plasmid that differed from the original pUC18 by either a 9 base pair (accepting state) or 11 base pair insert (unaccepting state) within the lacZ alpha region gene. Upon transformation and expression of the resultant plasmids in E. coli, the accepting state was represented by production of functional beta-galactosidase and formation of blue colonies on X-gal medium. In contrast, the unaccepting state was represented by white colonies due to a shift in the open reading frame of lacZ.

Assessing Combinations of Cytotoxic Agents Using Leukemia Cell Lines

The mainstay of clinical anti-neoplastic chemotherapy is multi-agent combinations, most of which were developed empirically. To speed research and decrease costs, there is increasing interest in moving new drugs into clinical trials in potentially active combinations based upon pre-clinical testing data. Because testing drug combinations in animals is expensive and complex, defining drug combinations initially in cell culture assays is essential. For in vitro testing we employ a panel of well-characterized cell lines and DIMSCAN, a semi-automatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable [using fluorescein diacetate (FDA)] cell numbers in tissue culture multi-well-plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by use of digital image processing and chemical quenching of fluorescence in non-viable cells with eosin Y. Cytotoxicity measured by DIMSCAN was found to be comparable to manual trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, that enables drug combination studies used to detect synergistic or antagonistic interactions in cell lines from both solid tumors and leukemias. While different mathematical models have been proposed for evaluating drug interactions, which can be classified as synergistic (combinations demonstrating greater than the additive activity expected from each agent alone), additive, or antagonistic (drugs showing less activity in combination than expected from the sum of each agent alone), we generally find the Combination Index method (as developed by Chou, et al.) to be the most suitable for evaluating of drug interactions in cell culture assays.