Honours in Biochemistry/Biotechnology/Chemistry
Abstracts of projects completed in 2007 and mid-2008
The Effects of Academic Stress in Human Subjects on the
Prevalence of Lactobacillus acidophilus and Streptococcus
mutans in Saliva Samples using RT-PCR
Harjinder Rayat
This study examined the impact of academic stress
on prevalence of salivary cortisol concentrations and activity
of Streptococcus mutans and Lactobacillus acidophilus in saliva
samples obtained from previous a study conducted by Knowles
et al., 2007. Saliva samples from 14 selected participants,
from a two week period; during the beginning of semester (non-stress
week) and during the first week of exams (high stress condition)
were collected. PCR successfully detected L. acidophilus,
however, failed to detect S. mutans. Therefore, L. acidophilus
was subjected to RT-PCR. Average cortisol levels (measured
from the previous study by Knowles et al., 2007), and starting
quantity (SQ) of DNA of L. acidophilus, in saliva samples
tested, were subjected to a paired samples t-test and Pearson’s
product-moment correlations. There was a reduction in levels
of L. acidophilus and an increase in cortisol levels from
non-stress week to stress week; L. acidophilus (M=2.86×104,
SD=1.01×105 versus M=3.38×102, SD=8.40×102),
and cortisol levels (M=4.98, SD=2.68 versus M=6.79, SD=3.35).
However, this difference was only significant for cortisol
levels t(13)=-3.05, p<0.01. In non-stress week and stress
week, there was a weak non-significant negative relationship
between L. acidophilus and cortisol levels. It could be possible
that other underlying factors, aside from levels of cortisol,
may have contributed to the decrease in L. acidophilus in
the non-stress week to stress week, although this decrease
was not significant. Future studies could concentrate on measuring
other hormones in the human body that could impact changes
in oral microflora.
Critical Factors in Microbiologically Induced Copper Corrosion.
David Lloyd
In this project involving the determination of critical
factors in copper corrosion it was found that factors in Blue-green
water (BGW) were responsible for accelerated corrosion. The
microbiological component of the BGW was implicated as the cause
of the accelerated corrosion. Due to observations of BGW inoculation
it was suspected that establishment of biofilm by particular
bacteria was critical to the MIC mode of action on copper. It
was proposed that characterisation of the bacteria involved
in the process, in particular the ability to form a biofilm,
the nature of the biofilm and the determination of its effect
on copper corrosion would be important in understanding the
corrosion.
Improving the Ethanol Tolerance of the Ethanol-Producing
Yeast Schizosaccharomyces pombe
Rebecca Peart
The aim of the project was to improve the ethanol tolerance
of the yeast Schizosaccharomyces pombe, using CSIRO’s
“Evolver” technology. This was to be achieved by
continuous selection, growing the yeast in increasing concentrations
of ethanol. The improvement in ethanol tolerance was to be demonstrated
by the ability of the evolved strain to grow at a higher concentration
of ethanol than the parent strain. In attempt to identify the
mutations responsible for improved ethanol tolerance, the sequence
of six ethanol tolerance related genes were to be compared between
the parent and evolved strain. Method development was prolonged,
involving altering the media to be suitable for growth of the
strain, and obtaining the desired conditions in the Evolver,
which proved more complex than anticipated. 18S sequencing was
also performed, verifying the identity of the strain. After
growing the yeast in the Evolver, five isolates were obtained.
The sequence of one ethanol tolerance gene was determined for
the parent strain, but no genes were sequenced for any of the
five isolates. The parent strain and the five isolates were
grown in a range of ethanol concentrations, but improved ethanol
tolerance was not demonstrated.
The Occurrence of Legionella Species in Recreational Waters
in Melbourne CBD
Barbara Vu
The present study aimed to evaluate the occurrence
of Legionella species in different locations within Melbourne’s
urban waterways. To assess the occurrence of Legionella species
in recreational waters in Melbourne CBD, standard microbiological
methods and molecular methods were used: isolation of Legionella
species using the standard culture method and PCR. Identification
of presumptive Legionella species was performed using Microbact
identification and gene sequencing. Results showed Legionella
species were detected in the Yarra river and L. pneumophila
was detected in Albert Park Lake. In addition, the detection
and identification of Legionella species and Legionella pneumophila
in recreational waters using molecular methods was more accurate
than using standard microbiological methods. Overall, the traditional
culture method used to detect Legionella species was found to
have low specificity compared to PCR techniques. Also, the molecular
techniques and phylogenetic analysis in combination with biochemical
identification tests are preferred for identification of Legionella
species.
The development of an innovative sterilisation technique
for biomaterial used in heart valve replacement surgery
Yury Shamis
The present study examined a range of conditions using
microwave radiation that could lead to the inactivation of bacteria
in transplant biomaterial. Two common pathogenic species of
bacteria; Escherichia coli and Staphylococcus aureus were used
as test microorganisms. The study initially examined the mechanical
durability of bovine pericardium under temperature stresses
in order to determine its limitations when applied to MW treatment.
The predicted change in mechanical functionality of bovine pericardium
treated using glutaraldehyde was also examined for confirmation.
Results showed that the mechanical functionality of bovine pericardium
was unaltered after it was heated to temperatures ranging from
40-70°C. Increased rigidity of glutaraldehyde treated pericardium
was also re-confirmed. The second part of the study experimentally
determined the parameters of high frequency MW radiation, at
sub lethal temperatures to bacteria, which would lead to successful
sterilisation of the bovine pericardium. Results showed that
at sub-lethal temperatures repeated exposure using high frequency
MW radiation at the optimised settings was significantly more
effective in decontaminating bovine pericardium of bacteria
compared to a single exposure. It was concluded that non thermal
inactivation of pathogenic bacteria in transplant biomaterial
could be achieved using high frequency MW radiation.
Baculovirus Expression of a Secreted Plant Protein
Rachel Dawson
Baculoviruses are rod-shaped dsDNA viruses found mainly
in insects. The most common baculovirus used for protein expression
is Autographa californica a multiple nuclear polyhedrosis
virus (AcNPV). It has been shown that chitinase is present within
the endoplasmic reticulum during baculovirus infection reducing
the levels of secreted recombinant protein produced. Deletion
of the chitinase and cathepsin genes has been claimed to increasing
the yield of secreted protein. The protease inhibitor precursor
from ornamental tobacco Nicotina alata (NaPI) is a serine protease
inhibitor, these inhibitors are thought play a role in protecting
plants from pests and pathogens. The aim of the project was
to test the efficiency of secretion of the NaPI protein using
a chitinase and cathepsin negative baculovirus at the GP64 and
polyhedrin locus. Two parallel approaches to remove the chitinase
and cathepsin genes from the baculovirus AcNPV were taken. The
first approach was to use a polyhedron locus based expression
given by the commercial Bacmagic system. A parallel attempt
was made to devise a system to effect recombination at the gp64
locus which would at the same time remove the chitinase and
cathepsin genes. A recombinant baculovirus was produced, RDNaPI,
with a chitinase and cathepsin deletion in its genome. The baculovirus
contained the NaPI gene under the control of a polyhedrin promoter
at the polyhedrin locus. The chitinase and cathepsin genome
deletions in the RDNaPI virus did not demonstrate greater protein
secretion of the protease inhibitor NaPI when compared to a
chitinase and cathepsin positive baculovirus. However there
appeared to be an increase in the integrity of the NaPI protein.
A transfer vector to produce a chitinase and cathepsin negative
recombinant baculovirus was tested but NaPI secretion from the
gp64 locus was not able to be completed.
A Study of the Kinetic Uptake of a Modified Baculovirus
Theo Xanthopoulos
Baculoviruses are double stranded DNA viruses that commonly
infect insects. Baculovirus glycoprotein gp64 is a major component
of the budded virion and mediates both binding and cell entry.
Bac283R9, a recombinant mutant, contains a 9-arginine oligopeptide
inserted into the gp64 open reading frame. While the mutant
virus is viable, it propagates to only low titres in cell culture.
This project set out to determine if the low titre of this virus
is a result of the slower adsorption to cells. A novel quantitative
PCR method was developed to enable virus to be measured.
Determining the effects of reduced gp64 was undertaken by comparing
the uptake kinetics of Bac283R9 with that of wild type gp64.
The Bac283R9 was observed to have a half life that was approximately
double that of the wild type gp64. Reduced adsorption rate to
cells may be a result of there being less gp64 on the surface
of virion.
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