Replica Plating Research Articles

Robot-assisted phenotyping of a Corynebacterium strain library towards valorization of next-generation feedstocks

The identification of microorganisms capable of efficiently using the different carbon sources (sugars, sugar acids, and sugar alcohols) and tolerating inhibitory by-products in hydrolysates is a challenge for the development of efficient bioproduction from lignocellulosic biomass (LCB). In this study, a robot-assisted screening approach was developed to characterize a collection of 32 Actinomycetes type strains, mainly belonging to the genus Corynebacterium. Using robot-assisted replica plating, the use of 12 different carbon-sources as well as the level of tolerance against prominent inhibitors derived from LCB was assessed. Based on a combined analysis of substrate utilization profiles and inhibitor tolerance five strains were chosen for further characterization in a miniaturised cultivation system using ultrafiltrated spent sulfite liquor (SSL) as the sole substrate. Noteworthy, in these experiments Corynebacterium sp. ATCC 14747 showed fast growth and biomass accumulation even in presence of high SSL concentrations.

The role of cytolethal distending toxin in Glaesserella parasuis JS0135 strain infection: Cytotoxicity, phagocytic resistance and pathogenicity

Glaesserella parasuis is an important porcine pathogen that commonly colonizes the upper respiratory tract of pigs and is prone to causing Glässer’s disease under complex conditions. As yet, the disease has led to serious economic losses to the swine industry worldwide. Studies so far have found that several virulence factors are associated with the pathogenicity of G. parasuis, but the pathogenic mechanism is still not fully understood. Cytolethal distending toxin (CDT), a potential virulence factor in G. parasuis, is involved in cytotoxicity, serum resistance, adherence to and invasion of host cells in vitro. Here, to further investigate the pathogenic role of CDT during G. parasuis infection in vitro and in vivo, a double cdt1 and cdt2 deletion mutant (Δcdt1Δcdt2) without selectable marker was first generated in G. parasuis JS0135 strain by continuous natural transformations and replica plating. Morphological observation and lactate dehydrogenase assay showed that the Δcdt1Δcdt2 mutant was defective in cytotoxicity. Additionally, the Δcdt1Δcdt2 mutant was more susceptible to phagocytosis caused by 3D4/2 macrophages compared to the wild-type JS0135 strain. Moreover, by focusing on clinical signs, necropsy, bacterial recovery and pathological observation, we found that the deletion of cdt1 and cdt2 genes led to a significant attenuation of virulence in G. parasuis. Taken together, these findings suggest that as an important virulence factor, CDT can significantly affect the pathogenicity of G. parasuis.

FORMULATION AND EVALUATION OF POMEGRANATE PEEL LOZENGES FOR MDR

The pomegranate fruits (Punica granatum) outer skin is the source of pomegranate peel extract (PPE). Thanks to its scientifically proven antibacterial, antioxidant, anticancer, antiulcer, and anti-inflammatory qualities, this extract has become more and more popular. This review concentrates on PPEs antibacterial efficacy against viruses, fungi, bacteria, and parasites that are resistant to many drugs. The serial dilution method and the cup plate method were used to test PSEs antibacterial activity. According to the findings, PPE exhibits a broad range of antibacterial properties against a variety of microorganisms, such as viruses (S. aureus, E. coli, K. pneumoniae, Salmonella, Salmonella enterica, P. aeruginosa, S. marcescens, Brucella spp., and R. glutinis), fungi, and mold (F. sambucinum, P. digitatum, Saccharomyces cerevisiae, Monilinialaxa, M. fructigena,B. coagulans, B. cereus, and B. subtilis). KEY WORDS: Pomegranate peel, Multi Drug Resistance, Antimicrobial Activity, Replica plate, Mutant Bacteria, Cytoplasmic membrane disruption.

The efficacy of ammonium as seed priming agents for promoting tomato (<i>Solanum lycopersicum</i> L.) germination under salinity stress

The aim of the current study was to assess the effectiveness of NH<sub>4+</sub> priming to enhance tomato seed germination and plant growth under saline stress. In the absence of light at 25 ± 1 °C, tomato seeds were primed with 50 and 100 mmol of NH<sub>4</sub>NO<sub>3</sub> and with 50 and 100 mmol of (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> for 12 and 24 h, respectively. In the seed germination test, twenty primed seeds were used for each treatment, along with five replica plates and a control, and were all incubated at 25°C with 25 mL of moistened water. In a second greenhouse experiment, primed seeds were planted in garden soil and watered with tap water, salt water, and a control. The concentration of saline water (50, 100, 150, and 200 mmol NaCl) was gradually raised after a 10-day break. The germination percentages in T5 (50 mmol (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 12 hrs) were 82±3.7% and 80±4.47 %, respectively, followed by T1 and T3 (50 mmol (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 12 h and 100 mmol NH<sub>4</sub>NO<sub>3</sub>, 12 h). When compared to the unprimed tomato seeds, the NH<sub>4+</sub> priming with NH<sub>4</sub>NO<sub>3</sub> and (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> improved plant height and other growth parameters. Furthermore, the chlorophyll and total flavonoid content were improved in both saline and non-saline treatments. In terms of salinity, the NH<sub>4+</sub> priming increased the proline content while decreasing the total protein content. It is concluded that further research will be needed to clarify the effect of NH<sub>4</sub>NO<sub>3</sub> and (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> as NH<sub>4+</sub> priming in tomato plants because other factors and nutrition can play a role in seed germination and plant growth development.

Killing of a Multispecies Biofilm Using Gram-Negative and Gram-Positive Targeted Antibiotic Released from High Purity Calcium Sulfate Beads.

Multispecies biofilm orthopedic infections are more challenging to treat than mono-species infections. In this in-vitro study, we aimed to determine if a multispecies biofilm, consisting of Gram positive and negative species with different antibiotic susceptibilities could be treated more effectively using high purity antibiotic-loaded calcium sulfate beads (HP-ALCSB) containing vancomycin (VAN) and tobramycin (TOB) in combination than alone. Three sets of species pairs from bioluminescent strains of Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) and clinical isolates, Enterococcus faecalis (EF) and Enterobacter cloacae were screened for compatibility. PA + EF developed intermixed biofilms with similar cell concentrations and so were grown on 316L stainless steel coupons for 72 h or as 24 h agar lawn biofilms and then treated with HP-ALCSBs with single or combination antibiotics and assessed by viable count or bioluminescence and light imaging to distinguish each species. Replica plating was used to assess viability. The VAN + TOB bead significantly reduced the PA + EF biofilm CFU and reduced the concentration of surviving antibiotic tolerant variants by 50% compared to single antibiotics. The combination of Gram-negative and positive targeted antibiotics released from HP-ALCSBs may be more effective in treating multispecies biofilms than monotherapy alone.

A replica plating method for efficient, high-throughput screening of antibiotic gene clusters in bacteria uncovers a holomycin-like cluster in the clinical isolate, Pantoea agglomerans 20KB447973

Bacterial natural products remain a major untapped source for novel antimicrobial scaffolds. Many of these products are encoded by biosynthetic gene clusters (BGCs), which can be identified using functional genomics. We developed a replica-plating approach to quickly screen for antibiotic production mutants from transposon mutant libraries and identify candidate antibiotic BGCs. In this technique, filter paper is used to transfer up to 200 mutants simultaneously onto a soft agar overlay or spread plate containing a target microbe to identify antibiotic-production mutants. These mutants can then be analyzed to identify disrupted genes and antibiotic BGCs. We first tested and optimized this technique by screening for previously characterized BGCs in Pantoea. We then applied the technique to uncover the gene cluster responsible for the production of an unknown broad-spectrum antibiotic from P. agglomerans 20KB447973, which we call Pantoea Natural Product 5 (PNP-5). Analysis of the predicted gene cluster for PNP-5 showed similarity to previously identified gene clusters for the broad-spectrum dithiolopyrrolone antibiotic, holomycin. Analysis of the spectrum of activity of PNP-5 showed activity against members of the Enterobacteriaceae, Erwiniaceae, and Streptococcaceae, including clinically relevant pathogens such as Klebsiella sp. and Escherichia coli. We also identified the production of a second antibiotic, pantocin A. Our findings demonstrate the utility of our replica-plating mutant transfer method in exploring unknown antibiotic BGCs. Adoption of this technique may accelerate the identification of potentially novel antimicrobial BGCs within strain collections, advancing the search for novel antimicrobials that can be used to treat multi-drug resistant infections.

The metagenomics of soil bacteria and fungi and the release of mechanical dormancy in hard seeds.

Persistence in the soil is a function of seed physiology, particularly non-germination and inherent lifespan. However, for seeds with mechanical dormancy, non-germination is also a function of the composition and activity of the soil microbiota. We attempted to screen out microorganisms in the soil that can specifically and rapidly decompose the hard fruit pericarps of Tilia miqueliana Maxim., a unique native tree species in China. Using the classical replica plating method, more than 100 different culturable microorganisms that could rapidly erode the pericarp were collected from the surface of pericarps under different culture conditions. At the same time, we successfully extended the concept of metagenomics and applied it to the identification of mixed artificial cultures. The decomposition process of the pericarps in soil was also simulated artificially. The physical and chemical data suggested a potential mechanism of microbial scarification and cracking in pericarp, whilst the embryos inside the eroded fruits retained good viability. Our discoveries could pave the way for the removal of physical and mechanical obstacles that prevent hard coat seeds from germinating. We anticipate that the use of this technology will improve the germination of other hard coat seeds. More research is needed to investigate the impacts on other seeds. The findings of this research can inform the design of experiments on the seed ecology of persistence.

Pressure tube replica imaging system for PHWR reactors based on optical coherence tomography

This paper describes a 3D profiling system for surface profiling of replicas obtained from pressure tube (PT) of PHWR/CANDU type reactors. This system is based on Optical Coherence Tomography (OCT) technique. Fiber based Fourier domain OCT configuration was used to design and develop the system considering the potential application of imaging radioactive PT replica samples. The sample scanning assembly of the system has been custom designed considering the cylindrical nature of PT replicas. Replica of inner surface of pressure tube was obtained on a replica plate using a custom built replica tool. Three PT replicas of non-radioactive pressure tubes with various flaws were obtained in different conditions. These replicas were imaged in 3D for identification and quantification of the flaws. This 3D profiling system termed as ‘PT replica imaging system’ is a promising tool which can be useful in periodic inspection of pressure tubes for safe operation of nuclear reactor.

The Discovery of Oropharyngeal Microbiota with Inhibitory Activity against Pathogenic Neisseria gonorrhoeae and Neisseria meningitidis: An In Vitro Study of Clinical Isolates.

With increasing incidence of pathogenic Neisseria infections coupled with emerging resistance to antimicrobials, alternative approaches to limit the spread are sought. We investigated the inhibitory effect of oropharyngeal microbiota on the growth of N. gonorrhoeae and N. meningitidis and the impact of the essential oil-based mouthwash Listerine Cool Mint® (Listerine). Oropharyngeal swabs from 64 men who have sex with men (n = 118) from a previous study (PReGo study) were analysed (ClinicalTrials.gov, NCT03881007). These included 64 baseline and 54 samples following three months of daily use of Listerine. Inhibition was confirmed by agar overlay assay, and inhibitory bacteria isolated using replica plating and identified using MALDI-TOF. The number of inhibitory isolates were compared before and after Listerine use. Thirty-one pharyngeal samples (26%) showed inhibitory activity against N. gonorrhoeae and/or N. meningitidis, and 62 inhibitory isolates were characterised. Fourteen species belonging to the genera Streptococci and Rothia were identified. More inhibitory isolates were observed following Listerine use compared to baseline, although this effect was not statistically significant (p = 0.073). This study isolated and identified inhibitory bacteria against pathogenic Neisseria spp. and established that daily Listerine use did not decrease their prevalence. These findings could provide a new approach for the prevention and treatment of pharyngeal Neisseria infections.

A novel and improved selective media for the isolation and enumeration of Klebsiella species

Bacterial pathogens are fostered in and transmitted through wastewater. Hence, monitoring their impact on sanitation and hygiene is imperative. As part of the monitoring process, culture-based methodologies are primarily used, which centre on the use of selective and differential media. Media available today are, at best, difficult to formulate and, at worst, prohibitively expensive. To address this lacuna, the study proposes a selective and differential medium for Klebsiella spp. Klebsiella blue agar (KBA) is completely selective against selected gram-positive bacteria (Bacillus spp., Staphylococcus aureus) and a few gram-negative bacteria (Acinetobacter baumanii, Serratia marcescens). On the other hand, it supports the growth of the chosen members of the Klebsiella pneumoniae species-complex with a characteristic green colouration. Methylene blue, tryptophan, and bile salt make up the selective components of KBA. Moreover, methylene blue, 0.6% NaCl, and glycerol render it differential. KBA was more selective than HiCrome™ Klebsiella Selective Agar Base (KSA) in replica plating experiments. KBA promoted only 157 CFUs against 209 CFUs in KSA when stamped with 253 CFUs grown on LB. The colonies so isolated were predominantly Klebsiella spp., on identification through colony polymerase chain reaction. Moreover, the differential nature of KBA distinguished Klebsiella aerogenes from other species. On the contrary, KSA lodged colonies indistinguishable from each other and Klebsiella spp. Due to its ease of formulation, high selectivity, differential nature, and cost-effective composition, KBA is a viable option for the routine culture of Klebsiella spp. in environmental and clinical settings.Key points• Formulated a novel selective and differential media for Klebsiella spp., named Klebsiella Blue agar• Facile formulation methodology• Can be employed to isolate Klebsiella spp. from complex sources such as wastewater

High-Throughput Screening for Thermostable Polyester Hydrolases.

Due to the promise of more sustainable recycling of plastics through biocatalytic degradation, the search for and engineering of polyester hydrolases have become a thriving field of research. Furthermore, among other methods, halo formation assays have become popular for the detection of polyester-hydrolase activity. However, established halo-formation assays are limited in their ability to screen for thermostable enzymes, which are particularly important for efficient plastic degradation. The incubation of screening plates at temperatures above 50°C leads to cell lysis and death. Therefore, equivalent master plates are commonly required to maintain and identify the active strains found on the screening plates. This replica plating procedure necessitates 20- to 60-fold more plates than our method, assuming the screened library is transferred to 384-well microtiter plates or 96-well microtiter plates, respectively, to organize the colonies in a retraceable manner, thus significantly lowering throughput. Here, we describe a halo formation assay that is designed to screen thermostable polyesterases independent of master plates and colony replication, thereby markedly reducing the workload and increasing the throughput.

Basic Bacteriological Routines.

In experimental bacteriology, bacteria are generally manipulated, stored, and shipped in the form of cultures. Depending on various factors, including strain genotype, storage and shipping methods, and manipulator skills, the culture may contain genetic variants or simply contaminants. It is therefore important to begin an experiment by streaking the culture on an agar plate. Streaking, a technique to disperse bacterial cells on the surface of the agar, serves the purpose of isolating individual colonies. A colony originates from a single cell and is a nearly pure culture. On rich LB medium after 24 h of incubation at 37°C, a colony of Salmonella contains ∼5 × 108 cells (about 29 generations). Streaking is also required in experiments that themselves generate single colonies as a result of selection (e.g., when constructing strains or introducing plasmids). Except in the few instances in which the selection efficiently kills all counter-selected bacteria, colonies growing on the selective plates are contaminated, sometimes heavily, with cells from the bacterial lawn. "Purifying" the colonies arising in such experiments by streaking on selective plates is therefore a mandatory step. Here, we show how this can be conveniently done using simple toothpicks. We also briefly describe the steps involved in inoculating liquid cultures, spreading plates, and replica plating.

Development of Live Attenuated Salmonella Typhimurium Vaccine Strain Using Radiation Mutation Enhancement Technology (R-MET).

Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.

Identification and characterisation of a Novel SXT/R391 ICE mobile genetic element isolated from an Irish wastewater environment

Human and animal pathogenic bacteria are constantly being released into the environment through human activity. Many of these organisms can harbour genes such as virulence genes, antibiotic resistance and heavy metal resistance genes that are inserted into plasmids, transposons and Integrating Conjugating elements (ICEs), leading to potential spread. Such spread can be detected among water and soil communities and in particular in wastewater treatment plants. This makes wastewater and treatment plants a potential reservoir for mobile genetic elements including SXT/R391 ICEs, commonly detected amongst enterobacterial genera. Many plasmid and ICE genomes have been detected serendipitously from clinical sources but few have been identified without selection. Here we examined a domestic wastewater treatment plant to identify, isolate and characterize SXT/R391 ICE’s without selection. Standard microbial replica plating in conjunction with ICE specific (conserved integrase gene) PCR techniques were employed to identify an SXT/R391 ICE MGE using a range of enterobactial selective media. A Novel SXT/R391 ICE MGE was identified from a wastewater Proteus mirabilisstrain. Whole genome sequencing using Ilumina sequencing technology revealed a novel 81 kb element which on annotation contained 75 open reading frames. The “hotspot regions”, which contain adaptive genes, encoded a novel bacteriophage defence mechanisms but lacked other selectable determinants. With the continuous arms race between bacteria and phage, bacteria have developed novel resistance mechanism systems that protect the bacteria from phage. Such systems may be key adaptive mechanisms harboured by ICEs particularly in wastewater systems which will contain large phage populations.

Bacteria-targeted fluorescence imaging of extracted osteosynthesis devices for rapid visualization of fracture-related infections

PurposeFracture-related infection (FRI) is a serious complication in orthopedic trauma surgery worldwide. Especially, the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to obtain proof-of-principle for the use of bacteria-targeted fluorescence imaging to detect FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery.MethodsExtracted osteosynthesis devices from 13 patients, who needed revision surgery after fracture treatment, were incubated with a near-infrared fluorescent tracer composed of the antibiotic vancomycin and the fluorophore IRDye800CW (i.e., vanco-800CW). Subsequently, the devices were imaged, and vanco-800CW fluorescence signals were correlated to the results of microbiological culturing and to bacterial growth upon replica plating of the imaged devices on blood agar.ResultsImportantly, compared to culturing, the bacteria-targeted fluorescence imaging of extracted osteosynthesis devices with vanco-800CW allows for a prompt diagnosis of FRI, reducing the time-to-result from days to less than 30 min. Moreover, bacteria-targeted imaging can provide surgeons with real-time visual information on the presence and extent of infection.ConclusionHere, we present the first clinical application of fluorescence imaging for the detection of FRI. We conclude that imaging with vanco-800CW can provide early, accurate, and real-time visual diagnostic information on FRI in the clinical setting, even in the case of low-grade infections.Graphical abstract

Assessing Antibiotic Tolerance of Staphylococcus aureus Derived Directly from Patients by the Replica Plating Tolerance Isolation System (REPTIS).

Antibiotic-tolerant Staphylococcus aureus poses a great challenge to clinicians as well as to microbiological laboratories and is one reason for treatment failure. Antibiotic-tolerant strains survive transient antibiotic exposure despite being fully susceptible in vitro. Thus, fast and reliable methods to detect tolerance in the routine microbiology laboratory are urgently required. We therefore evaluated the feasibility of the replica plating tolerance isolation system (REPTIS) to detect antibiotic tolerance in Staphylococcus aureus isolates derived directly from patients suffering from different types of infections and investigated possible connections to clinical presentations and patient characteristics. One hundred twenty-five S. aureus isolates were included. Replica plating of the original resistance testing plate was used to assess regrowth in the zones of inhibition, indicating antibiotic tolerance. Bacterial regrowth was assessed after 24 and 48 h of incubation, and an overall regrowth score (ORS) was assigned. Regrowth scores were compared to the clinical presentation. Bacterial regrowth was high for most antibiotics targeting protein synthesis and relatively low for antibiotics targeting other cellular functions such as DNA replication, transcription, and cell wall synthesis, with the exception of rifampin. Isolates with a blaZ penicillinase had lower regrowth in penicillin and ampicillin. Low ORSs were more prevalent among isolates recovered from patients with immunosuppression or methicillin-resistant S. aureus (MRSA) isolates. In conclusion, REPTIS is useful to detect antibiotic tolerance in clinical microbiological routine diagnostics. Further studies should evaluate the impact of rapid detection of antibiotic tolerance as a clinical decision-making tool for tailored antibiotic treatments.

Abundance of Multiple-Antibiotic-Resistant Salmonella Strains in Fecal Samples of Rhinoceros unicornis of the Kaziranga National Park, India

Aims: This study was undertaken to assess the abundance of multiple-antibiotic-resistant (MAR) Salmonella strains in fecal samples of Rhinoceros unicornis of the Kaziranga National Park (KNP), India.
 Study Design: Antibiotic-resistance profile of the Salmonella isolates from fecal samples of rhinoceros was carried out by replica plating on Muller Hinton Agar (MHA) plates containing antibiotics. The presence of class 1 integrons in metallo-β-lactamase (MBL) producing Salmonella isolates was determined by multiplex PCR.
 Place and Duration of Study: The study was carried out on rhinos of KNP situated in the Golaghat district of Assam, India in April 2015.
 Methodology: Fresh rhino fecal samples (designated as 1R, 2R, 3R, 4R and 5R) were dilution plated onto MacConkey agar. Purified bacterial colonies were then streaked separately on bismuth sulphite (BS) agar plates. All black colonies which are characteric growth of Salmonella were selected and used to make master plates on Luria Agar. To determine the antibiotic-resistance profile of the isolates, master-plates of purified single colonies of Salmonella spp. were replicate-printed on plates containing antibiotics from the β-lactam, aminoglycoside, or quinolone groups. To detect the presence of an integron, a conserved segment polymerase chain reaction was used.
 Results: 97.6% of the Salmonella isolates tested were resistant to >1 antibiotics (multidrug resistant or MAR). A total of 100 isolates from two fecal samples, 4R and 5R, were found to be imipenem resistant; 52 of them tested positive for the presence of MBLs. Five of the twenty MBL producing strains contained class 1 integrons.
 Conclusion: Because Salmonella is usually spread by drinking contaminated water, it is likely that water bodies in KNP are contaminated with MAR Salmonella strains. In case of Salmonellosis outbreak among Indian one-horned rhinos, our antibiogram will assist the veterinarians to choose the appropriate regimen of antibiotics for the rhinos in the KNP.

Stability of plasmid pBR322 within Escherichia coli cells

nsertion of plasmids into the bacterial cells is of great significance especially in course of the transfer of drug resistance, virulence and other traits. Retention of plasmids within the host bacteria is therefore an important factor for bacterial homeostasis. Current study inferred the pBR322 plasmid stability within the Escherichia coli competent cells. The calcium chloride heat shock method was used for the transformation purpose. The plasmid retention phenomenon was assessed through the replica plating. The results positively showed the plasmid retention within E. coli.

Isolation and Characterization of Lipid-Producing Bacillus sp. TC14

In order to obtain microbes producing microbial oil from environment, a strain, marked as TC14 was isolated by replica plating and Sudan Black staining on the selected media. Its colony is smooth; milk white, slightly transparent, round edge, sticky. Its cell is long-rod, G-, intracellular lipid granule. The results of physiological and biochemical research: catalase, denitrification and fluorescent pigment tests are negative; metachromatic granule staining, amylolysis, lecithinase tests are positive. According to its sequencing result of 16S rDNA, strain TC14 is classified and belonged to Bacillus, namely sp. TC14. The optimum inoculation temperature is 35°C and the most suitable initial pH value is 10. The intracellular lipid of strain TC14 is extracted by the way of hydrochloric acid hydrolysis and the calculated ratio of oil-production is 5.08%.

A New Method for Converting Aptamers to Riboswitches Using the Theophylline Riboswitch

Fluorescence Activated Cell Sorting, or FACS, is a method that was used to convert the theophylline aptamer into a riboswitch. This method could theoretically be used to convert other discovered aptamers into riboswitches, however it is a costly method and is only available to those with access to these high‐tech, expensive machines. The theophylline riboswitch was previously discovered by implementing the theophylline aptamer, with random sequences linking it to the expression platform, into a specifically‐designed plasmid, and then using a FACS machine to sort the cells.We can structure a new system that would select only the correct sequence, containing the theophylline riboswitch out of a pool, without the use of a FACS machine. To do so, we place the pool of sequences containing the aptamer, linked to the Shine Dalgarno by eight randomized nucleotides, into a newly designed plasmid, placNEO, and transform the plasmid into bacteria cells. Then, the use of replica plating along with screening will select the cells that only contain the plasmid with the correct riboswitch sequence. By doing so, we confirm that this system is efficient in converting aptamers into riboswitches without the need for a FACS machine.After an aptamer has been successfully converted into its riboswitch, the system of ratiometric fluorescence will allow for testing of the riboswitch’s function. This is done by designing a plasmid, pTRFlac, that contains genes for red and green fluorescence proteins, mCherry and GFP respectively, on either side of the inserted riboswitch. A PCR product encoding for mCherry, the riboswitch, and GFP will be inserted downstream of the lactose operon in pUC18. Ratios of the fluorescence intensities of the two florescent proteins will provide the ability to measure the riboswitch’s function through fluorescence readings.Support or Funding InformationMonmouth University Summer Research Program ICFNJ