Development of a high-level gene expression system for the production of bioplastics in sugarcane

Despite various approaches, the production of biodegradable plastics such as polyhydroxybutyrate (PHB) in transgenic plants has met with limited success due largely to low expression levels. Even in the few instances where high levels of protein expression have been reported, the transgenic plants have been stunted indicating PHB is phytotoxic (Poirier 2002). This PhD describes the application of a novel virus-based gene expression technology, termed InPAct („In Plant Activation.), for the production of PHB in tobacco and sugarcane. InPAct is based on the rolling circle replication mechanism by which circular ssDNA viruses replicate and provides a system for controlled, high-level gene expression. Based on these features, InPAct was thought to represent an ideal system to enable the controlled, high-level expression of the three phb genes (phbA, phbB and phbC) required for PHB production in sugarcane at a preferred stage of plant growth. A Tobacco yellow dwarf virus (TbYDV)-based InPAct-phbA vector, as well as linear vectors constitutively expressing phbB and phbC were constructed and different combinations were used to transform tobacco leaf discs. A total of four, eight, three and three phenotypically normal tobacco lines were generated from discs transformed with InPAct-phbA, InPAct-phbA + p1300-TaBV P-phbB/phbC- 35S T, p1300-35S P-phbA-NOS T + p1300-TaBV P-phbB/phbC-35S T and InPAct-GUS, respectively. To determine whether the InPAct cassette could be activated in the presence of the TbYDV Rep, leaf samples from the eight InPActphbA + p1300-TaBV P-phbB/phbC-35S T plants were agroinfiltrated with p1300- TbYDV-Rep/RepA. Three days later, successful activation was indicated by the detection of episomes using both PCR and Southern analysis. Leaf discs from the eight InPAct-phbA + p1300-TaBV P-phbB/phbC-35S T transgenic plant lines were agroinfiltrated with p1300-TbYDV-Rep/RepA and leaf tissue was collected ten days post-infiltration and examined for the presence of PHB granules. Confocal microscopy and TEM revealed the presence of typical PHB granules in five of the eight lines, thus demonstrating the functionality of InPActbased PHB production in tobacco. However, analysis of leaf extracts by HPLC failed to detect the presence of PHB suggesting only very low level expression levels. Subsequent molecular analysis of three lines revealed low levels of correctly processed mRNA from the catalase intron contained within the InPAct cassette and also the presence of cryptic splice sites within the intron. In an attempt to increase expression levels, new InPAct-phb cassettes were generated in which the castorbean catalase intron was replaced with a synthetic intron (syntron). Further, in an attempt to both increase and better control Rep/RepA-mediated activation of InPAct cassettes, Rep/RepA expression was placed under the control of a stably integrated alc switch. Leaf discs from a transgenic tobacco line (Alc ML) containing 35S P-AlcR-AlcA P-Rep/RepA were supertransformed with InPAct-phbAsyn or InPAct-GUSsyn using Agrobacterium and three plants (lines) were regenerated for each construct. Analysis of the RNA processing of the InPAct-phbAsyn cassette revealed highly efficient and correct splicing of the syntron, thus supporting its inclusion within the InPAct system. To determine the efficiency of the alc switch to activate InPAct, leaf material from the three Alc ML + InPAct-phbAsyn lines was either agroinfiltrated with 35S P-Rep/RepA or treated with ethanol. Unexpectedly, episomes were detected not only in the infiltrated and ethanol treated samples, but also in non-treated samples. Subsequent analysis of transgenic Alc ML + InPAct-GUS lines, confirmed that the alc switch was leaky in tissue culture. Although this was shown to be reversible once plants were removed from the tissue culture environment, it made the regeneration of Alc ML + InPAct-phbsyn plant lines extremely difficult, due to unintentional Rep expression and therefore high levels of phb expression and phytotoxic PHB production. Two Alc ML + InPAct-phbAsyn + p1300-TaBV P-phbB/phbC-35S T transgenic lines were able to be regenerated, and these were acclimatised, alcohol-treated and analysed. Although episome formation was detected as late as 21 days post activation, no PHB was detected in the leaves of any plants using either microscopy or HPLC, suggesting the presence of a corrupt InPAct-phbA cassette in both lines. The final component of this thesis involved the application of both the alc switch and the InPAct systems to sugarcane in an attempt to produce PHB. Initial experiments using transgenic Alc ML + InPAct-GUS lines indicated that the alc system was not functional in sugarcane under the conditions tested. The functionality of the InPAct system, independent of the alc gene switch, was subsequently examined by bombarding the 35S Rep/RepA cassette into leaf and immature leaf whorl cells derived from InPAct-GUS transgenic sugarcane plants. No GUS expression was observed in leaf tissue, whereas weak and irregular GUS expression was observed in immature leaf whorl tissue derived from two InPAct- GUS lines and two InPAct-GUS + 35S P-AlcR-AlcA P-GUS lines. The most plausible reason to explain the inconsistent and low levels of GUS expression in leaf whorls is a combination of low numbers of sugarcane cells in the DNA replication-conducive S-phase and the irregular and random nature of sugarcane cells bombarded with Rep/RepA. This study details the first report to develop a TbYDV-based InPAct system under control of the alc switch to produce PHB in tobacco and sugarcane. Despite the inability to detect quantifiable levels of PHB levels in either tobacco or sugarcane, the findings of this study should nevertheless assist in the further development of both the InPAct system and the alc system, particularly for sugarcane and ultimately lead to an ethanol-inducible InPAct gene expression system for the production of bioplastics and other proteins of commercial value in plants.

Development of the sustainable building and construction products industry in Australia

This paper draws on a major study the authors conducted for the Australian Government in 2009. It focuses on the diffusion issues surrounding the uptake of sustainable building and construction products in Australia. Innovative sustainable products can minimise the environmental impact during construction, while maximising asset performance, durability and re-use. However, there are significant challenges faced by designers and clients in the selection of appropriate sustainable products in consideration of the integrated design solution, including overall energy efficiency, water conservation, maintenance and durability, low-impact use and consumption. The paper is a review of the current state of sustainable energy and material product innovations in Australia. It examines the system dynamics surrounding these innovations as well as the drivers and obstacles to their diffusion throughout the Australian construction industry. The case product types reviewed comprise: solar energy technology, small wind turbines, advanced concrete technology, and warm-mixed asphalt. The conclusions highlight the important role played by Australian governments in facilitating improved adoption rates. This applies to governments in their various roles, but particularly as clients/owners, regulators, and investors in education, training, research and development. In their role as clients/owners, the paper suggests that government can better facilitate innovation within the construction industry by adjusting specification policies to encourage the uptake of sustainable products. In the role as regulators, findings suggest governments should be encouraging the application of innovative finance options and positive end-user incentives to promote sustainable product uptake. Also, further education for project-based firms and the client/end users about the long-term financial and environmental benefits of innovative sustainable products is required. As more of the economy’s resources are diverted away from business-as-usual and into the use of sustainable products, some project-based firms may face short-term financial pain in re-shaping their businesses. Government policy initiatives can encourage firms make the necessary adjustments to improve innovative sustainable product diffusion throughout the industry.

Development of the rural statistical sustainability framework tool

It is important to promote a sustainable development approach to ensure that economic, environmental and social developments are maintained in balance. Sustainable development and its implications are not just a global concern, it also affects Australia. In particular, rural Australian communities are facing various economic, environmental and social challenges. Thus, the need for sustainable development in rural regions is becoming increasingly important. To promote sustainable development, proper frameworks along with the associated tools optimised for the specific regions, need to be developed. This will ensure that the decisions made for sustainable development are evidence based, instead of subjective opinions. To address these issues, Queensland University of Technology (QUT), through an Australian Research Council (ARC) linkage grant, has initiated research into the development of a Rural Statistical Sustainability Framework (RSSF) to aid sustainable decision making in rural Queensland. This particular branch of the research developed a decision support tool that will become the integrating component of the RSSF. This tool is developed on the web-based platform to allow easy dissemination, quick maintenance and to minimise compatibility issues. The tool is developed based on MapGuide Open Source and it follows the three-tier architecture: Client tier, Web tier and the Server tier. The developed tool is interactive and behaves similar to a familiar desktop-based application. It has the capability to handle and display vector-based spatial data and can give further visual outputs using charts and tables. The data used in this tool is obtained from the QUT research team. Overall the tool implements four tasks to help in the decision-making process. These are the Locality Classification, Trend Display, Impact Assessment and Data Entry and Update. The developed tool utilises open source and freely available software and accounts for easy extensibility and long-term sustainability.

Multiculturalism, education for sustainable development (ESD) and the shifting discursive landscape of social inclusion

Australia has a long and sometimes turbulent relationship with the migrant Other. This paper examines a component of this relationship via the window of contemporary multicultural policy. The paper begins with an analysis of the political and social conditions that enabled a national and bipartisan policy of multiculturalism to emerge as formalised federal policy during the late 1960s and early 1970s. The paper re-problematises the influences that helped shape Australia's articulation of race and ethnicity and argues that multiculturalism, within a post-September 11 environment, can no longer be framed solely within its traditional framework of social justice. The paper positions education for sustainable development (ESD) as an emerging discursive field that provides educators with an alternative road map for critiquing Australia's fluid relationship with the migrant Other. By linking the tenets of multiculturalism with ESD, this paper suggests pre-service teacher educators are presented with a productive, and at the same time politically palatable, means for regaining pedagogical traction for a semi-dormant agenda of social inclusion.

Rating counselor - client behavior in online counseling: Development and preliminary psychometric properties of the Counseling Progress and Depth Rating Instrument

Although there are widely accepted and utilized models and frameworks for nondirective counseling (NDC), there is little in the way of tools or instruments designed to assist in determining whether or not a specific episode of counseling is consistent with the stated model or framework. The Counseling Progress and Depth Rating Instrument (CPDRI) was developed to evaluate counselor integrity in the use of Egan's skilled helper model in online counseling. The instrument was found to have sound internal consistency, good interrater reliability, and good face and convergent validity. The CPDRI is, therefore, proposed as a useful tool to facilitate investigation of the degree to which counselors adhere to and apply a widely used approach to NDC

Dublin Docklands Development Authority architectural concept competition for a landmark tower/U2 studio

The U2 Tower competition entry involved the architectural design for a landmark office tower with associated head office for the world acclaimed rock band U2. The selected site for the office tower was located on the banks of the river Liffey, Dublin. The tower design was intended as a signifier or gateway to the docklands and the city itself. The proposed design incorporated a podium level for music retail and a media centre, a concourse level including cafeteria and outdoor areas as well as a commercial tower.

An assessment of development funding for new housing post GFC in Queensland, Australia

Purpose: The purpose of this paper is to identify changes in bank lending criteria due to the GFC and to explore the associated impacts on new housing supply in Queensland, Australia. Design/methodology/approach: This research involves a survey of each of Australia’s big four banks, as well as two prominent arrangers of development finance. Data on key lending criteria was collected: Pre GFC, during the GFC, and GFC recovery stage. Findings: The GFC has resulted in a retraction of funds available for residential development. The few institutions lending are filtering out only the best credit risks by way of constrictive loan covenants including: low loan to value ratios, high cash equity requirements, regional “no go” zones, and demonstrated borrower track record. The ability of developers to proceed with new housing developments is being constrained by their inability to obtain sufficient finance. Research limitations/implications: This research uses survey data, together with an understanding of the project finance process to extrapolate impacts on the residential development industry across Queensland. No regional or sub-market analysis is included. Future research will include subsequent surveys to track any loosening of credit policies over time and sub-market sector analysis. Practical implications: The inability to obtain project finance is identified as a key constraint to new housing supply. This research will inform policy makers and provide important quantitative evidence of the importance of availability of development finance in the housing supply chain. Social implications: Queensland is facing a supply shortfall, which if not corrected, may lead to upward pressure on house prices and falling housing affordability. Originality/value: There is very little academic research on development funding. This research is unique in linking bank lending criteria to new housing supply and demonstrating the impact on the development industry.

The development of an observation tool for use with parents with psychiatric disability and their preschool children

Community-based treatment and care of people with psychiatric disabilities has meant that they are now more likely to engage in the parenting role. This has led to the development of programs designed to enhance the parenting skills of people with psychiatric disabilities. Evaluation of these programs has been hampered by a paucity of evaluation tools. This study's aim was to develop and trial a tool that examined the parent-child interaction within a group setting, was functional and easy to use, required minimum training and equipment, and had acceptable levels of reliability and validity. The revised tool yielded a single scale with acceptable reliability. It had discriminative validity and concurrent validity with non-independent global ratings of parenting. Sensitivity to change was not investigated. The findings suggest that this method of evaluating parenting is likely to have both clinical and research utility and further investigation of the psychometric properties of the tool is warranted.

Development and evaluation of the Clinician Suicide Risk Assessment Checklist

This paper describes the development and evaluation of a new instrument - the Clinician Suicide Risk Assessment Checklist (CSRAC). The instrument assesses the clinician's competency in three areas: clinical interviewing, assessment of specific suicide risk factors, and formulating a management plan. A draft checklist was constructed by integrating information from 1) literature review 2) expert clinician focus group and 3) consultation with experts. It was utilised in a simulated clinical scenario with clinician trainees and a trained actor in order to test for inter-rater agreement. Agreement was calculated and the checklist was re-drafted with the aim of maximising agreement. A second phase of simulated clinical scenarios was then conducted and inter-rater agreement was calculated for the revised checklist. In the first phase of the study, 18 of 35 items had inadequate inter-rater agreement (60%>), while in the second phase, using the revised version, only 3 of 39 items failed to achieve adequate inter-rater agreement. Further evidence of reliability and validity are required. Continued development of the CSRAC will be necessary before it can be utilised to assess the effectiveness of risk assessment training programs.

Development of a performance model for international construction joint venture : a system dynamics approach

The uncertain and dynamic nature of International Construction Joint Venture (ICJV) performance is evolved with many critical factors which lead to make partner relationships more complex in respect of making decisions to maintain a cohesive environment. Addressing to the fact, a generic system dynamics performance model for ICJV is developed by integrating a number variables as to get an overall impact on performance of ICJV and to make effective decisions based on that. In order to formulate and validate the model both structurally and behaviourally, both qualitative and quantitative data are gathered by conducting intensive interviews from two ICJVs in Thailand. After conducting intensive simulations of model, three major problems are identified related to negative value gap, low productivity in construction and high rate of ineffective information sharing of both ICJVs. Several policies are suggested and integrated application of these policies provides a maximum improvement to performance of the ICJV.

Measuring the effectiveness of computer-based scientific visualisations for conceptual development in Australian chemistry classrooms

Visual modes of representation have always been very important in science and science education. Interactive computer-based animations and simulations offer new visual resources for chemistry education. Many studies have shown that students enjoy learning with visualisations but few have explored how learning outcomes compare when teaching with or without visualisations. This study employs a quasi-experimental crossover research design and quantitative methods to measure the educational effectiveness - defined as level of conceptual development on the part of students - of using computer-based scientific visualisations versus teaching without visualisations in teaching chemistry. In addition to finding that teaching with visualisations offered outcomes that were not significantly different from teaching without visualisations, the study also explored differences in outcomes for male and female students, students with different learning styles (visual, aural, kinesthetic) and students of differing levels of academic ability.

Spatial Sciences Student Awards Recognition : News from School of Urban Development, QUT

Summary of Spatial Sciences (Surveying) Student Prize Ceremony were recently held at The Old Government House - QUT Cultural Precinct. This short industry article briefly outlines the 15 student award descriptions and some photos of 2011 recipients and thanks industry sponsors.

Neuropsychological development in children with early and continuously treated phenylketonuria : association with biochemical markers

PKU is a genetically inherited inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase. The failure of this enzyme causes incomplete metabolism of protein ingested in the diet, specifically the conversion of one amino acid, phenylalanine, to tyrosine, which is a precursor to the neurotransmitter dopamine. Rising levels of phenylalanine is toxic to the developing brain, disrupting the formation of white matter tracts. The impact of tyrosine deficiency is not as well understood, but is hypothesized to lead to a low dopamine environment for the developing brain. Detection in the newborn period and continuous treatment (a low protein phe-restricted diet supplemented with phenylalanine-free protein formulas) has resulted in children with early and continuously treated PKU now developing normal I.Q. However, deficits in executive function (EF) are common, leading to a rate of Attention Deficit Hyperactivity Disorder (ADHD) up to five times the norm. EF worsens with exposure to higher phenylalanine levels, however recent research has demonstrated that a high phenylalanine to tyrosine ratio (phenylalanine:tyrosine ratio), which is hypothesised to lead to poorer dopamine function, has a more negative impact on EF than phenylalanine levels alone. Research and treatment of PKU is currently phenylalanine-focused, with little investigation of the impact of tyrosine on neuropsychological development. There is no current consensus as to the veracity of tyrosine monitoring or treatment in this population. Further, the research agenda in this population has demonstrated a primary focus on EF impairment alone, even though there may be additional neuropsychological skills compromised (e.g., mood, visuospatial deficits). The aim of this PhD research was to identify residual neuropsychological deficits in a cohort of children with early and continuously treated phenylketonuria, at two time points in development (early childhood and early adolescence), separated by eight years. In addition, this research sought to determine which biochemical markers were associated with neuropsychological impairments. A clinical practice survey was also undertaken to ascertain the current level of monitoring/treatment of tyrosine in this population. Thirteen children with early and continuously treated PKU were tested at mean age 5.9 years and again at mean age 13.95 years on several neuropsychological measures. Four children with hyperphenylalaninemia (a milder version of PKU) were also tested at both time points and provide a comparison group in analyses. Associations between neuropsychological function and biochemical markers were analysed. A between groups analysis in adolescence was also conducted (children with PKU compared to their siblings) on parent report measures of EF and mood. Minor EF impairments were evident in the PKU group by age 6 years and these persisted into adolescence. Life-long exposure to high phenylalanine:tyrosine ratio and/or low tyrosine independent of phenylalanine were significantly associated with EF impairments at both time points. Over half the children with PKU showed severe impairment on a visuospatial task, and this was associated only with concurrent levels of tyrosine in adolescence. Children with PKU also showed a statistically significant decline in a language comprehension task from 6 years to adolescence (going from normal to subnormal), this deficit was associated with lifetime levels of phenylalanine. In comparison, the four children with hyperphenylalaninemia demonstrated normal function at both time points, across all measures. No statistically significant differences were detected between children with PKU and their siblings on the parent report of EF and mood. However, depressive symptoms were significantly correlated with: EF; long term high phe:tyr exposure; and low tyrosine levels independent of phenylalanine. The practice survey of metabolic clinics from 12 countries indicated a high level of variability in terms of monitoring/treatment of tyrosine in this population. Whilst over 80% of clinics surveyed routinely monitored tyrosine levels in their child patients, 25% reported treatment strategies to increase tyrosine (and thereby lower the phenylalanine:tyrosine ratio) under a variety of patient presentation conditions. Overall, these studies have shown that EF impairments associated with PKU provide support for the dopamine-deficiency model. A language comprehension task showed a different trajectory, serving a timely reminder that non-EF functions also remain vulnerable in this population; and that normal function in childhood does not guarantee normal function by adolescence. Mood impairments were associated with EF impairments as well as long term measures of phenylalanine:tyrosine and/or tyrosine. The implications of this research for enhanced clinical guidelines are discussed given varied current practice.

The evidence-based development of an intervention to address the information needs of adults newly diagnosed with primary brain tumours and their carers

Adults diagnosed with primary brain tumours often experience physical, cognitive and neuropsychiatric impairments and decline in quality of life. Although disease and treatment-related information is commonly provided to cancer patients and carers, newly diagnosed brain tumour patients and their carers report unmet information needs. Few interventions have been designed or proven to address these information needs. Accordingly, a three-study research program, that incorporated both qualitative and quantitative research methods, was designed to: 1) identify and select an intervention to improve the provision of information, and meet the needs of patients with a brain tumour; 2) use an evidence-based approach to establish the content, language and format for the intervention; and 3) assess the acceptability of the intervention, and the feasibility of evaluation, with newly diagnosed brain tumour patients. Study 1: Structured concept mapping techniques were undertaken with 30 health professionals, who identified strategies or items for improving care, and rated each of 42 items for importance, feasibility, and the extent to which such care was provided. Participants also provided data to interpret the relationship between items, which were translated into ‘maps’ of relationships between information and other aspects of health care using multidimensional scaling and hierarchical cluster analysis. Results were discussed by participants in small groups and individual interviews to understand the ratings, and facilitators and barriers to implementation. A care coordinator was rated as the most important strategy by health professionals. Two items directly related to information provision were also seen as highly important: "information to enable the patient or carer to ask questions" and "for doctors to encourage patients to ask questions". Qualitative analyses revealed that information provision was individualised, depending on patients’ information needs and preferences, demographic variables and distress, the characteristics of health professionals who provide information, the relationship between the individual patient and health professional, and influenced by the fragmented nature of the health care system. Based on quantitative and qualitative findings, a brain tumour specific question prompt list (QPL) was chosen for development and feasibility testing. A QPL consists of a list of questions that patients and carers may want to ask their doctors. It is designed to encourage the asking of questions in the medical consultation, allowing patients to control the content, and amount of information provided by health professionals. Study 2: The initial structure and content of the brain tumour specific QPL developed was based upon thematic analyses of 1) patient materials for brain tumour patients, 2) QPLs designed for other patient populations, and 3) clinical practice guidelines for the psychosocial care of glioma patients. An iterative process of review and refinement of content was undertaken via telephone interviews with a convenience sample of 18 patients and/or carers. Successive drafts of QPLs were sent to patients and carers and changes made until no new topics or suggestions arose in four successive interviews (saturation). Once QPL content was established, readability analyses and redrafting were conducted to achieve a sixth-grade reading level. The draft QPL was also reviewed by eight health professionals, and shortened and modified based on their feedback. Professional design of the QPL was conducted and sent to patients and carers for further review. The final QPL contained questions in seven colour-coded sections: 1) diagnosis; 2) prognosis; 3) symptoms and problems; 4) treatment; 5) support; 6) after treatment finishes; and 7) the health professional team. Study 3: A feasibility study was conducted to determine the acceptability of the QPL and the appropriateness of methods, to inform a potential future randomised trial to evaluate its effectiveness. A pre-test post-test design was used with a nonrandomised control group. The control group was provided with ‘standard information’, the intervention group with ‘standard information’ plus the QPL. The primary outcome measure was acceptability of the QPL to participants. Twenty patients from four hospitals were recruited a median of 1 month (range 0-46 months) after diagnosis, and 17 completed baseline and follow-up interviews. Six participants would have preferred to receive the information booklet (standard information or QPL) at a different time, most commonly at diagnosis. Seven participants reported on the acceptability of the QPL: all said that the QPL was helpful, and that it contained questions that were useful to them; six said it made it easier to ask questions. Compared with control group participants’ ratings of ‘standard information’, QPL group participants’ views of the QPL were more positive; the QPL had been read more times, was less likely to be reported as ‘overwhelming’ to read, and was more likely to prompt participants to ask questions of their health professionals. The results from the three studies of this research program add to the body of literature on information provision for brain tumour patients. Together, these studies suggest that a QPL may be appropriate for the neuro-oncology setting and acceptable to patients. The QPL aims to assist patients to express their information needs, enabling health professionals to better provide the type and amount of information that patients need to prepare for treatment and the future. This may help health professionals meet the challenge of giving patients sufficient information, without providing ‘too much’ or ‘unnecessary’ information, or taking away hope. Future studies with rigorous designs are now needed to determine the effectiveness of the QPL.

Development and validation of novel methods for microbial source tracking based on Escherichia coli as an indicator of water quality

Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.