Genotyping and subgenotyping of Trichophyton rubrum isolated from dermatophyt...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Genotyping and subgenotyping of Trichophyton rubrum isolated from dermatophyt...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
To Study the Genomic Fingerprinting of Relatedness in Strains of Bacillus Sp....ijtsrd
The aim of the present investigation was to study the genomic adaptation of Bacillus sp. isolated from chromium contaminated environment and normal environment. For this purpose ten highly chromium resistant Bacillus sp. CSB-1 to10 and ten normal Bacillus sp. NB-1 to 10 were isolated from soil samples collected from mine and its adjacent areas of Sukinda, Odisha. The genomic DNA was isolated from 20 Bacillus species and made free from contamination of RNA by RNAase treatment. The molecular weight of DNA from 20 Bacillus species were determined by comparing with DNA . The genomic variation study of 10 chromium resistant and 10 normal strains of Bacillus was done by RAPD analysis. Among the 10 primers used only two OPT 01 and OPT 05 gave the amplification. OPT 01 primer gave lesser bands in normal Bacillus species than those obtained in chromium resistant Bacillus species. The presence of those DNA fragment band in chromium resistant Bacillus can be used as a molecular marker to identify chromium resistant Bacillus from normal Bacillus. These chromium resistant Bacillus species after further assessment of their potential to reduce the toxic hexavalent form to its nontoxic trivalent form can be exploited for the bioremediation of toxic and carcinogenic soluble hexavalent chromium containing industrial effluents. Sasmita Das | Pratima Pradhan | Ajay Kumar Sahu "To Study the Genomic Fingerprinting of Relatedness in Strains of Bacillus Sp. by RAPD Analysis" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-2 , February 2019, URL: https://www.ijtsrd.com/papers/ijtsrd20284.pdf
Paper URL: https://www.ijtsrd.com/biological-science/biotechnology/20284/to-study-the-genomic-fingerprinting-of-relatedness-in-strains-of-bacillus-sp-by-rapd-analysis/sasmita-das
Isolation and characterization of a fungus for extracellular synthesis of sma...Nanomedicine Journal (NMJ)
Abstract
The use of biogenic selenium nanoparticles for various purposes is going to be an issue of considerable importance; thus, appropriate simple methods should be developed and tested for the synthesis and recovery of these nanoparticles. In this study, a fungus was isolated from a soil sample, identified as Aspergillus terreus and used for extracellular synthesis of selenium nanoparticles (Se NPs). UV–Vis spectroscopy and energy dispersive X-ray spectrum studies were carried out to confirm Se NPs formation within 60 min. Dynamic light scattering and scan electron microscopic methods were also used to characterize both size and shapes of the Se NPs. The results show that spherical particles with average size of 47 nm were formed by adding a culture supernatant of A. terreus to selenium ions solution. This approach appears to be an easy and appropriate method for extracellular synthesis of small Se NPs. Extracellular synthesis of small Se NPs has not been reported yet.
Abstract
Objective(s):
The development of reliable and ecofriendly process for the synthesis of nano-metals is an important aspect in the field of nanotechnology. Nano-metals are a special group of materials with broad area of applications.
Materials and Methods:
In this study, extracellular synthesis of silver nanoparticles (SNPs) performed by use of the gram positive soil Streptomycetes. Streptomycetes isolated from rice fields of Guilan Province, Iran (5 isolates). Initial characterization of SNPs was performed by visual change color. To determine the bacterium taxonomical identity, its colonies characterized morphologically by use of scanning electron microscope. The PCR molecular analysis of active isolate represented its identity partially. In this regard, 16S rDNA of isolate G was amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then conducted using NCBI GenBank database using BLAST. Also SNPs were characterized by, transmission electron microscopy (TEM) and X-ray diffraction spectroscopy (XRD).
Results:
From all 5 collected Streptomyces somaliensis isolates, isolate G showed highest extracellular synthesis of SNPs via in vitro. SNPs were formed immediately by the addition of (AgNO3) solution (1 mM). UV-visible spectrophotometry for measuring surface plasmon resonance showed a single absorption peak at 450 nm, which confirmed the presence of SNPs. TEM revealed the extracellular formation of spherical silver nanoparticles in the size range of 5-35 nm.
Conclusions:
The biological approach for the synthesis of metal nanoparticles offers an environmentally benign alternative to the traditional chemical and physical synthesis methods. So, a simple, environmentally friendly and cost-effective method has been developed to synthesize AgNPs using Streptomycetes.
The objectives of this study were to detect and characterize the phytoplasma in tissues of diseased hibiscus plants using Dains’ stain light microscopy and molecular based techniques. Molecular characterization was performed using the DNA sequencing and phylogenetic analysis of the spacer region between 16S and 23S rRNA fragment of the isolated phytoplasma genome. This work concerning phytoplasma associated witches' broom (group 16SrII) diseases of hibiscus plants is achieved for the first time in Egypt.
In vitro mutagenesis of Cymbidium La bell “Anna Belle” by γ-rays irradiation ...IJEAB
The optimum media for multiplication of protocorm like bodies (PLBs) and shoot buds of Cymbidium La bell “Anna Belle” were studied in order to prepare the in vitro samples for irradiation. The values of LD50 (lethal dose of 50% samples) of PLBs, shoot buds and plantlets of tested Cymbidium after cultivation of 4 months were also determined about 35.0, 41.0 and 83.1 Gy, respectively. The addition of oligochitosan played as an very important trigger for promotion on the generation of shoot bud from PLBs after irradiation. The in vitro variations have been generated by γ-rays irradiation of PLBs with doses in range of 20 - 50 Gy. The highest mutant frequency (3.83‰) of C. La bell was found by the irradiation of PLB samples at 30 Gy. The different properties of obtained in vitro variations compared to wild types were found to be chlorophyll, short leaves, long leaves, and violet pericardium variations. The genetic relationships among generated variant lines in M1V4 and wild type were analyzed using RAPD techniques.
Develop an understanding of Taxonomy (classification) of Oral Microorganisms
Describe how to obtain samples from Oral Cavity
Describe Molecular techniques of identification
Describe techniques that requires culture for identification
To Study the Genomic Fingerprinting of Relatedness in Strains of Bacillus Sp....ijtsrd
The aim of the present investigation was to study the genomic adaptation of Bacillus sp. isolated from chromium contaminated environment and normal environment. For this purpose ten highly chromium resistant Bacillus sp. CSB-1 to10 and ten normal Bacillus sp. NB-1 to 10 were isolated from soil samples collected from mine and its adjacent areas of Sukinda, Odisha. The genomic DNA was isolated from 20 Bacillus species and made free from contamination of RNA by RNAase treatment. The molecular weight of DNA from 20 Bacillus species were determined by comparing with DNA . The genomic variation study of 10 chromium resistant and 10 normal strains of Bacillus was done by RAPD analysis. Among the 10 primers used only two OPT 01 and OPT 05 gave the amplification. OPT 01 primer gave lesser bands in normal Bacillus species than those obtained in chromium resistant Bacillus species. The presence of those DNA fragment band in chromium resistant Bacillus can be used as a molecular marker to identify chromium resistant Bacillus from normal Bacillus. These chromium resistant Bacillus species after further assessment of their potential to reduce the toxic hexavalent form to its nontoxic trivalent form can be exploited for the bioremediation of toxic and carcinogenic soluble hexavalent chromium containing industrial effluents. Sasmita Das | Pratima Pradhan | Ajay Kumar Sahu "To Study the Genomic Fingerprinting of Relatedness in Strains of Bacillus Sp. by RAPD Analysis" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-2 , February 2019, URL: https://www.ijtsrd.com/papers/ijtsrd20284.pdf
Paper URL: https://www.ijtsrd.com/biological-science/biotechnology/20284/to-study-the-genomic-fingerprinting-of-relatedness-in-strains-of-bacillus-sp-by-rapd-analysis/sasmita-das
Isolation and characterization of a fungus for extracellular synthesis of sma...Nanomedicine Journal (NMJ)
Abstract
The use of biogenic selenium nanoparticles for various purposes is going to be an issue of considerable importance; thus, appropriate simple methods should be developed and tested for the synthesis and recovery of these nanoparticles. In this study, a fungus was isolated from a soil sample, identified as Aspergillus terreus and used for extracellular synthesis of selenium nanoparticles (Se NPs). UV–Vis spectroscopy and energy dispersive X-ray spectrum studies were carried out to confirm Se NPs formation within 60 min. Dynamic light scattering and scan electron microscopic methods were also used to characterize both size and shapes of the Se NPs. The results show that spherical particles with average size of 47 nm were formed by adding a culture supernatant of A. terreus to selenium ions solution. This approach appears to be an easy and appropriate method for extracellular synthesis of small Se NPs. Extracellular synthesis of small Se NPs has not been reported yet.
Abstract
Objective(s):
The development of reliable and ecofriendly process for the synthesis of nano-metals is an important aspect in the field of nanotechnology. Nano-metals are a special group of materials with broad area of applications.
Materials and Methods:
In this study, extracellular synthesis of silver nanoparticles (SNPs) performed by use of the gram positive soil Streptomycetes. Streptomycetes isolated from rice fields of Guilan Province, Iran (5 isolates). Initial characterization of SNPs was performed by visual change color. To determine the bacterium taxonomical identity, its colonies characterized morphologically by use of scanning electron microscope. The PCR molecular analysis of active isolate represented its identity partially. In this regard, 16S rDNA of isolate G was amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then conducted using NCBI GenBank database using BLAST. Also SNPs were characterized by, transmission electron microscopy (TEM) and X-ray diffraction spectroscopy (XRD).
Results:
From all 5 collected Streptomyces somaliensis isolates, isolate G showed highest extracellular synthesis of SNPs via in vitro. SNPs were formed immediately by the addition of (AgNO3) solution (1 mM). UV-visible spectrophotometry for measuring surface plasmon resonance showed a single absorption peak at 450 nm, which confirmed the presence of SNPs. TEM revealed the extracellular formation of spherical silver nanoparticles in the size range of 5-35 nm.
Conclusions:
The biological approach for the synthesis of metal nanoparticles offers an environmentally benign alternative to the traditional chemical and physical synthesis methods. So, a simple, environmentally friendly and cost-effective method has been developed to synthesize AgNPs using Streptomycetes.
The objectives of this study were to detect and characterize the phytoplasma in tissues of diseased hibiscus plants using Dains’ stain light microscopy and molecular based techniques. Molecular characterization was performed using the DNA sequencing and phylogenetic analysis of the spacer region between 16S and 23S rRNA fragment of the isolated phytoplasma genome. This work concerning phytoplasma associated witches' broom (group 16SrII) diseases of hibiscus plants is achieved for the first time in Egypt.
In vitro mutagenesis of Cymbidium La bell “Anna Belle” by γ-rays irradiation ...IJEAB
The optimum media for multiplication of protocorm like bodies (PLBs) and shoot buds of Cymbidium La bell “Anna Belle” were studied in order to prepare the in vitro samples for irradiation. The values of LD50 (lethal dose of 50% samples) of PLBs, shoot buds and plantlets of tested Cymbidium after cultivation of 4 months were also determined about 35.0, 41.0 and 83.1 Gy, respectively. The addition of oligochitosan played as an very important trigger for promotion on the generation of shoot bud from PLBs after irradiation. The in vitro variations have been generated by γ-rays irradiation of PLBs with doses in range of 20 - 50 Gy. The highest mutant frequency (3.83‰) of C. La bell was found by the irradiation of PLB samples at 30 Gy. The different properties of obtained in vitro variations compared to wild types were found to be chlorophyll, short leaves, long leaves, and violet pericardium variations. The genetic relationships among generated variant lines in M1V4 and wild type were analyzed using RAPD techniques.
Develop an understanding of Taxonomy (classification) of Oral Microorganisms
Describe how to obtain samples from Oral Cavity
Describe Molecular techniques of identification
Describe techniques that requires culture for identification
Microbial DNA extracted from two soil samples collected from Beni-Suef and Kafr El-Sheikh were
subjected to PCR amplification with primers specific for 16S rDNA gene and cloned in linear pCR 2.1
plasmid vector. Recombinants were transformed into Escherichia coli competent cells. Sixty clone
inserts (30 from each soil sample) were sequenced and subjected to phylogenetic analyses. Forty
sequences of the sixty clones were affiliated with previously recognized bacterial groups. Thirty six of
these had closest relatives among cultured taxa and clustered primarily with three divisions containing
microrganisms commonly associated with soil: Proteobacteria, Gram-positive organisms, and
Cytophaga-Flexibacter-Bacteroides group. The results also showed the presence of one clone related to
Nirospira retrieved from Beni-Suef soil, one clone from Archaea kingdom retrieved from Kafr El-Sheikh
soil, and three clones affiliated to the newly described Holophaga-Acidobacterium phylum in both Beni-
Suef and Kafr El-Sheikh soils. Seven sequences grouped with known divisions but had closest relatives
among soil taxa known only from rDNA sequences analysis. Twelve clone sequences were distantly
related to known sequences. Many of these sequences may represent new bacterial divisions.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Nucleic Acid-its structural and functional complexity.
Final research paper
1. 1
Isolation of a novel mycobacteriophage from the soils of Puerto Rico
Lizbeth Pérez Castro, and Javier Zavala Ayala
Biomedical Techniques Course, RISE Program,
Universidad de Puerto Rico, Recinto de Cayey
Bacteriophages are a class of virus. This specific class of virus infects only bacteria. The
bacteriophages cannot propagate outside their host bacteria. They’re composed of a protein
coat, which encloses its DNA or RNA, and a tail that provides anchorage. The purpose of this
investigation is to isolate a novel bacteriophage from the tropical soil of Puerto Rico. In order to
do so, there are four major steps namely: collection of the environmental sample, isolation of the
phage from the environmental sample, purification of the phage, and characterization of the
phage. Investigating phages is important because many phages can shed light not only on
viruses, but also on their host. Also the identification of the different phages can lead to
answering the question on how and why this organism keeps evolving. Understanding these two
factors can be transcendental for medical applications. Conclusions are that the purpose of the
investigation was achieved since the isolation of a novel mycobacteriophage was successfully
done from soil found in a cowshed in Gurabo, P.R.
Introduction:
Human beings often live without
thinking about all the microscopic organisms
that live among us. Bacteriophages are one of
many microorganisms, and they are a class of
virus. This specific class of virus infects only
bacteria. The bacteriophages cannot propagate
outside their host bacteria. Bacteriophages are
composed of a protein coat, which encloses
DNA or RNA, and a tail that provides
anchorage. The morphology of the
bacteriophages varies.
When a phage attaches to a bacterium,
it releases its genetic material into the host.
This genetic material combines with the hosts
genetic material and replicates. Afterwards
more phages are made and then released. There
are two types of phage cycles: lytic and
lysogenic. A key difference between the lytic
and lysogenic phage cycles is that in the lytic
phage, the viral DNA exists as a separate
molecule within the bacterial cell, and
replicates separately from the host bacterial
DNA. The location of viral DNA in the
lysogenic phage cycle is within the host DNA.
In both cases, the virus/phage replicates using
the host DNA machinery, but in the lytic phage
cycle, the phage is a free floating separate
molecule in the host DNA.
In this experiment, the bacterial hosts
used are Mycobacterium Smegmatis and
Bacillus. Mycobacteria are responsible for
many diseases and deaths worldwide (WHO
2010). It is estimated that one third of the
world's population are infected with
Mycobacterium Tuberculosis (Reyrat and
Kahnt 2001). But Mycobacterium Smegmatis is
a non-pathogenic bacterium and has a fast
growing rate, which makes them suitable for
laboratory experiments (Gordon and Smith
1953). It also acts as an aerobic organism.
Bacillus is an aerobic organism that can be
either a free-living or parasitic pathogenic
species. Bacillus and M. Smegmatis share some
common characteristics such as: rod-shaped
morphology and both are gram positive. The
hypothesis established was that new phages
will be isolated from tropical soils in Puerto
Rico.
The study of phages is important,
because they can be used as tools to move
DNA around for cloning and mutation. Genetic
2. 2
information about different phages allows
scientists to compare phages, study biodiversity
and identify new genes that may be useful for
scientific or therapeutic applications. Scientist
would like to use phages to kill specific
antibiotic-resistant bacteria that cause diseases
(Asai, 2012).
Materials and Methods:
For this experiment, soil samples were
taken in order to isolate a novel bacteriophages
from the tropical soil of Puerto Rico. The
climate of Puerto Rico influenced the
availability of bacteriophages, because phages
are mostly found in moist areas surrounded by
organic material (feces). There are four major
steps for the isolation of a bacteriophage, these
are: collection of the environmental sample,
isolation of the phage from the environmental
sample, purification of the phage, and
characterization of the phage.
For the isolation of a bacteria phage,
samples of soil were collected with the use of a
sterile spoon and sterile bag. Every sample
collection was identified by location, texture of
the soil, date, and time collected. The sample
was going to be tested with two host: Bacillus
cereus (B. cereus) and Mycobacterium
smegmatis (M. smegmatis). After three sample
collections, the soil sample located in Road
#943, Sector Los Chinos, Gurabo in the
grounds of a cowshed had phages. The
coordinates of the place are 18º 16’ 7’ N, 65º
58’ 32’ W. This last soil sample was collected
in the morning of a temperate or mild day next
to cow feces on February 24, 2014.
Approximately 0.5g of soil were weighted. To
one of the samples of 0.5 g of soil, 10 ml of the
master mix were added. To the other 0.5 g
sample of soil, 10 ml of TSB were added.
Then 1ml of B. cereus were added to the soil
containing the 10 ml of TSB, and 1 ml of M.
smegmatis were added to the soil mixed with
the 10 ml of master mix. These enrichments
were placed in the incubator at 37ºC, shaking at
220 revolutions per minute (rpm) for 24 hours.
The next day the sample was ready for
the next procedure, the isolation of the phage.
The enrichments were centrifuged for 10
minutes at 3000 rpm and 5 ml of the
supernatant was taken and filtrated with a 0.20
micrometers filter. The bacterial host was
identified. The plaque screening is done with
an isolated phage plug, and the phage
purification is completed with the use of the
streak protocol. These purification procedures
were repeated three times. Then after the third
purification, a second enrichment was done by
adding 10 ml of the master mix and 1 ml of M.
smegmatis to a phage plug of the third
purification petri dish. This enrichment was left
incubating at 37ºC, shaking at 220 rpm for a
time period of 24 hours.
The next step was to do eight dilutions
with an isolated phage plug of the third
purification. The spot test was done with these
dilutions. Then after identifying a dilution that
forms a web pattern, 80 microliters of the
identified dilution was mixed with 5 ml of M.
smegmatis. This second enrichment was then
divided equally and seeded in 9 plates. After
incubating for 24 hours at 37ºC, the web
pattern was observed. Five milliliters of phage
buffer were added to the nine plates with the
web pattern and incubated in the freezer at 2ºC.
The next day the plates were taken out and the
top agar was broken down in order to obtain all
the phages. The phages and buffer were taken
with a pipette and mixed in a 50 ml tube. This
mixture was centrifuged at 3000 rpm in the
time lapse of 10 minutes. The supernatant was
obtained and vacuum filtered. This last step
was done in order to obtain the High Titer
Phage Lysate (HTPL). With the HTPL a
second dilution was done first at -2,-4,-6,-7,-8,-
9 and the streak protocol followed. The phage
plugs done were counted per dilution. Then
proteomics were done in the workshop given
by Dr. Michael Rubin on April 25, 2014. On
this workshop 15 microliters of the HTPL and
15 microliters of Beta-mercaptoethanol (BME)
were mixed. Then 25 microliters of the mixture
were loaded on a polyacrylamide gel that went
from 12 to 1 percent. An EM grid was also
prepared in order to analyze later the phage
using Scanning Electron Microscopy. The dye
3. 3
used to stain the phage in the EM grid was 10
microliters of 1% uranyl acetate. The last
procedure done was the isolation of the phage
genomic DNA. To obtain the DNA 10ml of the
HTPL were prepared by adding 40 microliters
of Nuclease Mix and incubating it at 37ºC for
30 minutes. Then it was taken out and left for
an hour at room temperature. Four milliliters of
phage precipitant solution were added to the
mixture and left overnight at 4ºC. The next day
the mixture was centrifuge and spun at
10,000xg for 20 minutes. The supernatant was
decanted carefully without disturbing the pellet.
0.5 mL of sterile ddH2O and two milliliters of
pre-warmed (37ºC) DNA Clean Up Resin were
added and gently swirled to mix. The water-
resin-phage-genomic-DNA solution was
divided into two columns and by filtering and
centrifuging. The solutions that weren’t needed
were removed and the phage genomic DNA
was obtained and eluted together in one
column. In order to analyze the DNA an
agarose gel was prepared. The DNA was
prepared by adding 8 microliters of 1X TBE
buffer, 2 microliters of the DNA and 2
microliters of tracking dye. Ten microliters of
each solution were loaded in the gel and ran at
100V. The gel after about an hour was
observed.
Results:
After the enrichment, filtration, and
streaking of the specimen on the petri dishes,
positive results were not obtained from plates
one and two. However, the third soil sample
had positive results. The coordinates of the
location where that sample was found are 18º
16’ 7’ N, 65º 58’ 32’ W. The petri dish
displayed phage plaques on the first streak
region. The three purifications were done, and
after obtaining a phage plaque from the third
purification, the dilutions were made. The
dilution used to make the nine plates was the
dilution 10−1
. Since the top agar that day was
not enough to make the ten plates, nine were
done. After evaluating the nine petri dishes for
web patterns, the steps necessary to obtain the
HTPL were done. The HTPL had a volume of
35ml. For the last steps, new dilutions were
made to see the number of phage colonies per
liter. The results can be seen in table 2. Then
the median was calculated using the number of
phages divided by the dilution and volume
(ml). The median was 1.57 ± 0.3 x 1010
PFU/ml. On figure 4 the results of the
polyacrylamide gels can be observe.
Nitidusvenutus compared with other phages
show distinct band patterns. The results
obtained from the last procedure which was the
isolation of the DNA were negative. The
agarose gel shows two band patterns instead of
one indicating that there was contamination.
Soil Samples Information
Sample Coordinates Soil
Description
Sample
#1
18º 16’ 16’ N,
65º 58’ 10’ W
Loose, and
moist
Sample
#2
18º 6’ 58’ N,
66º 9’ 19’ W
Moist and
Chunky
Sample
#3
18º 16’ 7’ N,
65º 58’ 32’ W
Loose, and
moist next to
cow feces
Table 1. Description and location of the
different soil samples.
Number of Phage Colonies per Dilution
Dilution Number
of Phages
colonies
Photo
10−6 133
10−7 18
4. 4
10−8 1
Table 2. Presents the illustrated results of the
number of plaques obtain per dilutions. The
dilutions that are not illustrated had complete
lysis.
Figure 1. Petri dish shows positive results, this
picture shows the phage plaques found after
streaking the filtrated mixture of the third
sample of soil.
Figure 2. Shows the third purification.
Figure 3. Shows the High Titer Phage Lysate
obtained.
Figure 4. Results of the duplicate
polyacrylamide gels. Nitidusvenutus is placed
on the fifth well.
5. 5
Figure 5. Shows the results of the DNA
Agarose gel. Nitidusvenutus was loaded on the
fifth well. Two different bands appear in the
fifth well indicated that the isolation of the
phage DNA wasn’t successful.
Discussion:
Isolation of a novel bacteriophage is a task that
requires patience, time, and effort. In order to
effectively locate the phages three samples of
soil were collected. The results for the first two
samples were negative. The soil sample that
had possible results was moist and loose, and it
was near cow feces. Animals all have extensive
microbiomes, full of different species of
bacteria. It can be assumed that sampling in
areas rich in organic waste products of other
organisms can be an indicative of phages. This
is possible because maybe the host organism
was amongst this animal’s microbiota. The
phages that were in the environment now had
better possibilities of infecting the bacterium
once it was free in the feces. The phage
obtained was isolated and purified. Our
bacterial host was M. smegmatis. The dilution
that showed web pattern and was used to make
the nine plates was the dilution10−1
. Nine
plates were used to obtain the HTPL instead of
ten.
Throughout all the processes done,
aseptic technique is crucial to avoid
contamination. Our mycobacteriophage
Nitidusvenutus showed clear plaques meaning
that it probably has a lytic life cycle. The gel
obtained from the proteomic workshop showed
that Nitidusvenutus had distinct band patterns
in comparison with the other phages. Since
phages can be cluster in families by the
proteins in their capsid, the next step is to
identify the cluster that Nitidusvenutus belongs
or is related to. The results obtained from the
isolation of the DNA were negative. The
agarose gel showed two band patterns instead
of one indicating that in the process of the
preparation of the solution there was
contamination.
Many phages shed light not only on
viruses, but also on their host. The
identification of the different phages can lead to
answering the question on how and why this
organism keeps evolving. Understanding these
two factors can be transcendental for medical
applications. After performing the experiments
or procedures, the hypothesis was proven,
phages were successfully isolated from the
soils of Puerto Rico. The soils of Puerto Rico
contain novel bacteriophage that show genetic
variations as seen on the on the polyacrylamide
gel results. For future work we want to
characterize the phage structure through
Transmission Electron Microscopy, and
sequence its DNA using bioinformatics.
Acknowledgement:
Our special recognition to the
contributions of: Giovanni Cruz, Gustavo
Martínez, Edwin Alvarado, and Juan Apiz for
serving as technical assistants. Special thanks
to Dr. Eneida Díaz and Dr. Elena González, for
their roles as course professors of Biomedical
Techniques (BIOL-4997). Our gratitude to Dr.
Michael Rubin, for his role as mentor and the
RISE program and the Howard Hughes
Medical Institute, for the materials needed for
the procedures.
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