Glycerol Stocks Research Articles

Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period.

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3–20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

2’Fucosyllactose and Bifidobacterium longum subsp. infantis Supplementation Modulates Immune Development of Piglets

ObjectivesProbiotics and 2’fucosyllactose (2’FL) have been shown to affect immune development in infants, but less is known about their synbiotic administration. Herein, the effects of 2’FL and Bifidobacterium longum subsp. infantis (Bi-26) on immune development was investigated in the young pig. MethodsMale piglets (N = 53 were provided ad libitum access to milk replacer without (CON) or with 1g/L 2’FL (FL) from postnatal day 2 to 34/35. Pigs were then stratified to receive Bi-26 prepared in glycerol stock (109 CFU) or glycerol stock alone (BI and FLBI). Blood, mesenteric lymph nodes (MLN), and ascending colon (AC) and rectal (RC) contents were collected. MLN and peripheral blood mononuclear cells (PBMC) immune cells were quantified by flow cytometry. Immunoglobulins (Igs) were measured by ELISA. PBMC and MLN cells were stimulated ex vivo with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) for 72 h. Cytokines in serum and ex vivo cell supernatants were measured by multiplex assay. Cytokine data were analyzed by multivariate LCA-model. All other data were analyzed by a 2-way ANOVA with fixed effects of prebiotic and probiotic. ResultssIgA tended (P = 0.07) to be higher in RC than AC, with no treatment effect. Serum IgG and IgM and MLN and PBMC immune cells were unaffected by treatment. Serum, IL-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-10, IL-12 and IL-18 were all higher in FL than CON (p < 0.05). In unstimulated PBMC, IL-18 was lower in FL, BI and FLBI than CON (p < 0.05). In LPS-stimulated PBMCs, IFNγ was higher in FL and IL1-RA was higher in FLBI than CON (p < 0.05). LPS had no effect on MLN cytokines; but, PHA increased IL-17 in FL and FLBI versus CON (p < 0.05). ConclusionsCompared to CON, serum cytokines involved in Th1 T-cell differentiation, IL-2, IL-12 and IL-18 were higher in FL piglets. This was countered by the increased production of IL-4 and IL-10. IL-1RA also increased, potentially balancing higher IL-1α and IL-1β in FL piglets. These differences were not observed in FLBI. LPS stimulated IFNγ secretion by PBMC from FL, whereas cells from FLBI secreted more IL-1RA. These findings suggest that dietary 2’FL primes T-cells for proinflammatory cytokine secretion, which is modulated by co-administration of Bi-26. Funding SourcesDuPont Nutrition and Biosciences.

Effects of 2’fucosyllactose and Bifidobacterium longum subsp. infantis on the Brain and Cognitive Development in the Young Pig

ObjectivesPrebiotics and probiotics have been studied for their beneficial effects on various aspects of development, but much remains unknown regarding interactive effects of their supplementation. This study investigated the effects of dietary 2’fucosyllactose (2’FL; prebiotic) and Bifidobacterium longum subsp. infantis (Bi-26; probiotic) on behavioral and brain structural development in the young pig. MethodsFifty-two intact male pigs were provided ad libitum access to a nutritionally adequate milk replacer without (control) or with 0.532% 2’FL from postnatal day (PND) 2 to 34/35. Pigs were further stratified to receive either glycerol stock alone or Bi-26 (109 CFU) in glycerol stock solution daily. All pigs were subjected to the novel object recognition (NOR) task, a behavioral paradigm assessing recognition memory, from PND 27 to 31. Pigs underwent magnetic resonance imaging procedures at PND 32 or 33 to assess absolute and relative brain volume. Additionally, brain microstructure was quantified using diffusion tensor imaging (DTI) to assess fractional anisotropy, and axial, radial, and mean diffusivity. ResultsBehavioral outcomes confirmed that there were no effects of prebiotics or probiotics on recognition memory (P > 0.05). Interactive effects (P < 0.05) were observed for the number of object visits, latency to the first object visit, and number of sample object visits. Pigs that received the probiotic supplementation (P < 0.05) spent less time engaging with the sample object in total and on average, were observed to have smaller absolute brain volumes for 9 of 27 total observed regions of interest, and smaller relative volumes for 2 regions associated with kinesthesia. Differences in axial diffusivity were observed in pigs supplemented with the probiotic for 2 of the 12 regions assessed. Interactive effects (P < 0.05) were observed for all DTI outcomes for a variety of brain regions. Trelative volume for the pons was observed to be larger for pigs supplemented with the prebiotic. ConclusionsDietary supplementation of prebiotics and probiotics elicited interactive effects on several exploratory behaviors and structural brain components, and supplementation with probiotics appeared to reduce the volume of specific brain regions. Funding SourcesDuPont Nutrition & Biosciences.

1163. Epidemiology of Candidemia: Can Candida Spread from Patient to Patient in the Hospital?

BackgroundCandidemia has become an increasingly important infection in recent years. Antifungal drug resistance in non-albicans species of Candida is increasingly common. Recent global emergence of Candida auris is a concern, owing to person-to-person transmission and survival on fomites. Our study aimed to determine if hospital transmission of diverse Candida species is occurring similar to what is seen in Candida auris. We analyzed all candidemia infections for signatures of transmission, including species, geographical, and temporal clusters. Here we present our preliminary data from December 2019 - May 2020.MethodsThis is a prospective and retrospective analytical observational study. Patients with candidemia were identified with the help of the Clinical Microbiology Lab at a University Medical Center. Data was collected on all identified patients by retrospective chart review. Data was described in terms of frequency distributions and percentages, and analyzed using SPSS. Isolates have been stored prospectively as glycerol stocks at -80 C for ongoing analyses.Results37 patients were identified (Tables 1 and 2). Clusters of candidemia were seen in the months of January (C. parapsilosis, 3 patients), February (C.glabrata, 3 patients), March (C.albicans, 5 patients) and April (C.glabrata, 3 patients). 33/37 (89%) had a central line prior. Lines were removed in 73% (24/33) of these patients, the remaining patients were deceased before lines could be removed. Pancreatic pathology was seen in 15/37 (40.5%) patients (Table 3). 25/37 (67.5%) had an Ophthalmology consult.Table 1. Patient demographics Table 2. Epidemiology of candidemia Table 3. Pancreatic pathology in candidemia ConclusionIt is possible that the clusters identified shared equipment or other environmental factors that caused nosocomial transmission. We plan to use Whole Genome Sequencing to determine clonality among these isolates. The association of candidemia with pancreatic pathology was curious. It is to be evaluated whether this was simply a confounder or an actual risk factor that perhaps warrants consideration of prophylaxis. Rates of Ophthalmology consults to evaluate for endophthalmitis need to be improved in our setting. We hope that this study would prove valuable for infection control efforts and help us be better prepared to tackle emerging pathogens of this genus.Disclosures All Authors: No reported disclosures

High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang.

Multi-omics approaches, including metagenomics and single-cell amplified genomics, have revolutionized our understanding of the hidden diversity and function of microbes in nature. Even in the omics age, cultivation is an essential discipline in microbial ecology since microbial cultures are necessary to assess the validity of an in silico prediction about the microbial metabolism and to isolate viruses infecting bacteria and archaea. However, the ecophysiological characteristics of predominant freshwater bacterial lineages remain largely unknown due to the scarcity of cultured representatives. In an ongoing effort to cultivate the uncultured majority of freshwater bacteria, the most abundant freshwater Actinobacteria acI clade has recently been cultivated from Lake Soyang through catalase-supplemented high-throughput cultivation based on dilution-to-extinction. This method involves physical isolation of target microbes from mixed populations, culture media simulating natural habitats, and removal of toxic compounds. In this protocol, we describe detailed procedures for isolating freshwater oligotrophic microbes, as well as the essence of the dilution-to-extinction culturing. As a case study employing the catalase-supplemented dilution-to-extinction protocol, we also report a cultivation trial using a water sample collected from Lake Soyang. Of the 480 cultivation wells inoculated with a single lake-water sample, 75 new acI strains belonging to 8 acI tribes (acI-A1, A2, A4, A5, A6, A7, B1, B4, C1, and C2) were cultivated, and each representative strain per subclade could be revived from glycerol stocks. These cultivation results demonstrate that the protocol described in this study is efficient in isolating freshwater bacterioplankton harboring streamlined genomes.

Experimental Evolution of Antifungal Resistance in Cryptococcus neoformans.

Cryptococcus neoformans, an opportunistic yeast-like fungal pathogen, has demonstrated resistance to all major classes of antifungals used to treat cryptococcal meningitis. However, combatting this fungal disease is an ongoing challenge among clinicians due to the evolution of antifungal-resistant strains. The limited availability of clinically approved antifungals has heightened the urgency to investigate the molecular mechanisms underscoring resistance. Studying how a fungal pathogen evolves to an antifungal drug in vitro using experimental evolution provides a simple, yet powerful approach to study the mechanisms of antifungal resistance. Experimental evolution involves the serial passaging of microbial populations under laboratory conditions, such that adaptive mutations can occur and be monitored in real time. This technique plays a key role in investigating the mechanisms of antifungal resistance in C. neoformans, and this can help in developing novel strategies to combat the emergence of resistance. Here, we outline how to make overnight cultures of C. neoformans and how to perform experimental evolution, and we present a spectrophotometric analysis to evaluate the evolution of antifungal resistance. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth and sample preparation of Cryptococcus neoformans Basic Protocol 2: Experimental evolution of antifungal resistance Basic Protocol 3: Analyzing the evolution of antifungal resistance Basic Protocol 4: Glycerol stock preparation.

Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus and Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology. Traditionally, rescuing virus from an infectious cDNA clone requires propagating plasmids in bacteria, which can result in mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative- rolling circle amplification (RCA), an in vitro technology. We demonstrate that the viral yield of transfected RCA product is comparable to midiprepped plasmid, albeit with a slight delay in kinetics. RCA, however, is cheaper and less time-consuming. Further, sequential RCA did not introduce mutations into the viral genome, subverting the need for glycerol stocks and retransformation. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.

Agrobacterium tumefaciens-Mediated Transformation of Three Milkweed Species (Asclepias hallii, A. syriaca, and A. tuberosa: Apocynaceae).

Milkweeds have ecological significance for insect herbivores that rely on them as hosts for either part of or the entirety of their life cycles. Interesting interactions, some of which are not completely understood, have evolved over time. To develop these species as models to elucidate the interplay with insect herbivores, we established Agrobacterium tumefaciens-mediated transformation approaches for Asclepias hallii (Hall's milkweed), A. syriaca (common milkweed), and A. tuberosa (butterflyweed). The method is based on infection of stem internodal explants, which were more amenable to transformation than leaf explants. We found that addition of freshly prepared dithiothreitol was critical to prevent browning of stem explants. Depending on the species, the time from infection to the regeneration of transgenic lines ranges from 2 to 4 months. Transformation efficiency for A. hallii was 9%, whereas efficiencies for A. syriaca and A. tuberosa were 6% and 13%, respectively. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Agrobacterium tumefaciens-mediated transformation of Asclepias internodal stem explants Basic Protocol 2: Preparation of Agrobacterium glycerol stocks containing gene constructs.

Off-Colony Screening of Biosynthetic Libraries by Rapid Laser-Enabled Mass Spectrometry.

By leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering, the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS)-a form of ambient laser desorption ionization mass spectrometry-to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies within <4 h, while simultaneously generating recoverable glycerol stocks of each colony in real time. This showcases LA-REIMS as a prescreening tool to complement downstream quantification methods such as liquid chromatography-mass spectroscopy (LCMS). By prescreening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.

Factors affecting microbial metabolism in a human fecal fermentation model to evaluate prebiotics

In vitro fermentations using human fecal bacteria are an often used high through put strategy to evaluate the prebiotic potential of food ingredients. We investigated the influence of the substrate presented to fecal microbiota by monitoring the microbial short chain fatty acid production. We also examined the impact of cryopreserved glycerol stocks of feces on the performance of multiple batches of fermentations to examine the feasibility of using standardized inocula for multiple batches. Both composition and concentrations of bioactives (dietary fibre, polyphenols) were found to influence the metabolic capacity of human fecal microbiota. We also found that feces preserved at −80 °C provided a consistent and stable inoculum for performing batch fermentations for a duration of up to 3 years.

Molecular characterisation of antibiotic-resistant Salmonella enterica isolates recovered from meat in South Africa

Salmonella enterica is a leading cause of human gastroenteritis in both developed and developing countries, causing significant economic losses on humans and animals worldwide. There are several routes for contracting salmonellosis, but the consumption of contaminated foods is by far the most frequent cause of human infections. This study aimed to assess the prevalence and resistance determinants of S. enterica isolates obtained from meat. Swab samples from meat were cultured for the identification of Salmonella spp., and 239 presumptive S. enterica isolates were recovered, purified and kept in glycerol stocks. The S. enterica. confirmed by polymerase chain reaction (PCR) were further tested against 15 antimicrobials using the disc-diffusion method on Muller-Hinton agar and the genotypic antimicrobial resistance determinants by PCR. Resistance among tetracyclines, bêta-lactams, and 3rd generation cephalosporins was found to be most frequent with a noticeable rise in the number of multi-drug resistance ranging from two to seven antimicrobials. A total of 20 resistance determinants were assessed with their prevalence and distributions obtained as follows; [aminoglycosides: aadA (89%), aacC2 (100%), aphA1 (38%), aphA2 (0%) and strA (7%)], [β-lactams: (ampC 100%), blaTEM, (33%), blaZ (17%) and blaOXA (10%)], [Chloramphenicol: catI (9%), catII (7%), and cmIA1 (10%)] and [tetracyclines: tetA (57%), tetB (30%), tetC (11%), tetD (73%), tetK (20%) and tetM, (43%)], and [sulfonamides: sulI (82%), sulII (7%)]. The findings signify a high prevalence of multidrug-resistant (MDR) S. enterica isolates and resistance determinants indicating increased public health risks associated with the consumption of contaminated meat.

Comparison of pellicle and shake flask-grown BCG strains by quality control assays and protection studies

A global BCG vaccine shortage began in 2013 which impacted availability for infant vaccinations, as well as preclinical studies and clinical trials of new TB vaccines. Stakeholders met in 2015 at McGill University in Montreal to discuss the shortage and potential mitigation strategies. Manufacturing BCG through a more tractable liquid fermentation process instead of the traditional pellicle growth method was considered a potentially viable strategy. This pilot program compared pellicle-grown and shake flask-grown BCG strains (as a first step towards modeling fermenter-produced BCG vaccine) in selected quality control assays, as well as mouse and guinea pig protection studies. Conventional pellicle-grown, lyophilized BCG WHO Reference Reagents (Danish, Moreau, Russian, Tokyo strains) were obtained from the National Institute for Biological Standards and Control (NIBSC), UK. Strains were grown in shake flasks and glycerol stocks prepared. Shake flask-grown BCG culture preparations generally met the requirements of quality control testing at NIBSC. In mouse and guinea pig protection studies there were no significant differences in lung colony forming units (CFUs) between shake flask-grown and pellicle-grown preparations, with the exception of BCG Russian, where the shake flask-grown preparation protected better in mice (P = 0.0042), but the pellicle-grown preparation protected better in guinea pigs (P = 0.0015). Producing BCG vaccines by a more tractable liquid growth process could be a viable solution to the global BCG shortage.

Variability in Characterizing Escherichia coli from Cattle Feces: A Cautionary Tale.

Shiga toxin-producing Escherichia coli (STEC) are diverse bacteria, with seven serogroups (O26, O45, O103, O111, O121, O145, O157; “Top 7”) of interest due to their predominance in human disease. Confirmation of STEC relies on a combination of culturing, immunological and molecular assays, but no single gold standard for identification exists. In this study, we compared analysis of STEC between three independent laboratories (LAB) using different methodologies. In LAB A, colonies of Top 7 were picked after serogroup-specific immunomagnetic separation of feces from western-Canadian slaughter cattle. A fraction of each colony was tested by PCR (stx1, stx2, eae, O group), and Top 7 isolates were saved as glycerol stocks (n = 689). In LAB B, a subsample of isolates (n = 171) were evaluated for stx1 and stx2 using different primer sets. For this, approximately half of the PCR were performed using original DNA template provided by LAB A and half using DNA extracted from sub-cultured isolates. All Top 7 isolates were sub-cultured by LAB A and shipped to LAB C for traditional serotyping (TS) to determine O and H groups, with PCR-confirmation of virulence genes using a third set of primers. By TS, 76% of O groups (525/689) matched PCR-determined O groups. Lowest proportions (p < 0.05) of O group matches between PCR and TS (62.6% and 69.8%) occurred for O26 and O45 serogroups, respectively. PCR-detection of stx differed most between LAB A and LAB C. Excluding isolates where O groups by PCR and TS did not match, detection of stx1 was most consistent (p < 0.01) for O111 and O157:H7/NM. In contrast, for O45 and O103, stx1 was detected in >65% of isolates by LAB A and <5% by LAB C. Stx2 was only detected by LAB C in isolates of serogroups O121, O145, and O157:H7/NM. LAB B also detected stx2 in O26 and O157:H12/H29, while LAB A detected stx2 in all serogroups. Excluding O111 and O157:H7/NM, marked changes in stx detection were observed between initial isolation and sub-cultures of the same isolate. While multiple explanations exist for discordant O-typing between PCR and TS and for differences in stx detection across labs, these data suggest that assays for STEC classification may require re-evaluation and/or standardization.

Mating-based Overexpression Library Screening in Yeast.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

Lyophilization of Bdellovibrio bacteriovorus 109J for Long-Term Storage.

Bdellovibrio bacteriovorus 109J is a Gram-negative predatory bacterium with obligate host dependency on other Gram-negative bacteria. This bacteriolytic predator collides with, enters, and establishes growth within the prey (host) periplasm, eventually lysing the prey cell wall to release fresh, motile B. bacteriovorus progeny. Laboratory maintenance of B. bacteriovorus has been previously described by other investigators. The protocols included in this unit deal with the technique required to lyophilize or freeze dry host-dependent B. bacteriovorus. This is an alternative means to frozen glycerol stocks for the long-term storage of B. bacteriovorus. It includes the cultivation process and methods to lyophilize B. bacteriovorus as well as recommended storage conditions. In addition, this unit provides insight on the formulation's shelf-life including the time to active culture after reviving lyophilized stocks of B. bacteriovorus following short-, medium-, and long-term storage. © 2017 by John Wiley & Sons, Inc.

The involvement of the ceramide transporter in Alzheimer’s disease Investigating the binding properties of the ceramide transporter in the human brain by optimizing the splitubiquitin Yeast Two-Hybrid system

Background: Alzheimer’s disease (AD) is the most common form of dementia, characterized by aggregates of Amyloid-ß (Aβ) peptides. The ceramide transporter (CERTL ) exists in a complex with serum amyloid P component in the Aβ plaques of AD patients. The goal is to obtain more information about the protein binding activity of CERTL in the human brain. To be able to do so, the Yeast Two-Hybrid (Y2H) technique, that was used to confirm the results from a previously executed CERTL Y2H screening, had to be optimized. Technical problems were encountered with the plasmid purification of the yeast NMY51 using the EZNA plasmid DNA minikit I and with the transformation in competent E.coli cells. Initially it was believed that there was a problem with the EZNA plasmid DNA minikit I. In this report troubleshooting was performed to identify the problem of the plasmid purification technique that has been used. Methods: To examine the protein binding activity of CERTL a split-ubiquitin Y2H system was used. In this system proteins are attached to two parts of one complete ubiquitin molecule, called the split-ubiquitin. The middle region domain of CERTL (the bait) was attached to the C-terminal half of the split-ubiquitin and the transcription factor LexA-VP16. The possible interactor, a prey library containing human brain proteins was fused to the N-terminal half of the split-ubiquitin. The presence of an interaction between bait and prey will results in the re-assembly of the split-ubiquitin, which activates ubiquitin proteases. The transcription factor is released and reporter genes are activated.The results of a previously executed Y2H screening had to be confirmed by performing another Y2H screening. The new screening had to be carried out using glycerol stocks of the yeast NMY51, containing both bait and prey, from the earlier performed Y2H screening. To optimize prey plasmid purification from the yeast using the EZNA plasmid DNA minikit I, troubleshooting was performed on the technique. Next to this, other methods to isolate the plasmid DNA, a QIAprep spin miniprep kit protocol and a phenol extraction and ethanol precipitation, were performed. The failing results were possibly due to the yeast being stored as a glycerol stock. In order to correct for this, a freshly transformed yeast sample was made. Results: Extracting the plasmid from glycerol stocks from the yeast NMY51 did not succeed. Plasmid purification and transformation of the prey plasmid from the fresh transformed yeast in E.coli competent cells was successful. Conclusion: From the results it is now believed that storing the yeast NMY51 containing the prey plasmid in glycerol stocks causes the prey plasmid to integrate into the genomic DNA of the yeast. Performing a new Y2H screening would therefore only be possible on fresh transformed yeast NMY51 cells.

Methods for Long-Term Stable Storage of Colletotrichum Species.

In this chapter we describe methods for long-term preservation of ascomycete genus Colletotrichum species. Colletotrichum species employ a hemibiotrophic infection strategy and cause clear anthracnose diseases on various host plants including the model plant Arabidopsis thaliana. Their infection proceeds in a highly synchronized manner, which is helpful for the dissection of the fungus-plant interactions at the molecular level. Gene engineering methods, including efficient protocols for targeted gene disruption, and whole-genome sequences are available for several Colletotrichum species. Thus, these pathogens provide us with model systems to address the molecular mechanisms underlying hemibiotrophic fungal pathogenicity.We describe how to prepare glycerol stocks or filter paper fungal stocks for long-term preservation of Colletotrichum species. These two methods are easily handled, and provide a stable preservation for at least a few years.

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-(2)H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the (2)H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.