Questions for discussion for children, parents, schools,and churches

Compilation of animation worksheets (01 - 07) - Questions for discussion for children, parents, schools and churches. Faith in the Nexus - Animation and Discussion Booklet

The synthesis of novel antibacterial proteins in the Chlamydomonas reinhardtii chloroplast

The Chlamydomonas reinhardtii chloroplast represents an attractive platform for therapeutic protein production, not least due to a robust molecular toolkit, low cost of cultivation, and the lack of endotoxins or potentially infectious agents in the algal host. The primary focus of this thesis has been the expression of bacteriophage endolysins in the C. reinhardtii chloroplast. Endolysins hold great promise as antibacterials since they can induce lysis of specific bacterial pathogens without affecting the body’s natural flora, do not result in acquired resistance in the pathogen, and can kill pathogens that colonize mucosal surfaces and biofilms. The expression of the lysin cpl-1 specific to the human pathogen Streptococcus pneumoniae has been confirmed in the C. reinhardtii chloroplast. The enzyme has subsequently been purified, and its lytic activity against culture collection and clinical strains of S. pneumoniae demonstrated, both for crude and enriched extracts. Two further endolysins, gp20 (specific to Propionibacterium acnes, strongly associated with clinical acne vulgaris) and lys16 (specific to Staphylococcus aureus, a common hospital acquired infection), have failed to express to detectable levels. This has instigated new investigations into the various factors affecting foreign gene expression in the C. reinhardtii chloroplast. Research has been conducted both in a wet lab context (with the use of modified leader sequences, full protein fusions and overhauled gene design) and in silico (looking particularly at codon- and codon pair usage in a wide panel of endogenous and recombinant genes). A defined codon pair bias has been shown to be present in the C. reinhardtii chloroplast, the first such bias to be reported in any organelle. The codon preferences observed have been related to a panel of transgenes that have previously been introduced into the chloroplast in the Purton lab, although no correlation has been found between codon pair usage and transgene expression

Synthesis of bacteriophage lytic proteins against Streptococcus pneumoniae in the chloroplast of Chlamydomonas reinhardtii.

There is a pressing need to develop novel antibacterial agents given the widespread antibiotic resistance among pathogenic bacteria and the low specificity of the drugs available. Endolysins are antibacterial proteins that are produced by bacteriophage-infected cells to digest the bacterial cell wall for phage progeny release at the end of the lytic cycle. These highly efficient enzymes show a considerable degree of specificity for the target bacterium of the phage. Furthermore, the emergence of resistance against endolysins appears to be rare as the enzymes have evolved to target molecules in the cell wall that are essential for bacterial viability. Taken together, these factors make recombinant endolysins promising novel antibacterial agents. The chloroplast of the green unicellular alga Chlamydomonas reinhardtii represents an attractive platform for production of therapeutic proteins in general, not least due to the availability of established techniques for foreign gene expression, a lack of endotoxins or potentially infectious agents in the algal host, and low cost of cultivation. The chloroplast is particularly well suited to the production of endolysins as it mimics the native bacterial expression environment of these proteins while being devoid of their cell wall target. In this study the endolysins Cpl-1 and Pal, specific to the major human pathogen Streptococcus pneumoniae, were produced in the C. reinhardtii chloroplast. The antibacterial activity of cell lysates and the isolated endolysins was demonstrated against different serotypes of S. pneumoniae, including clinical isolates and total recombinant protein yield was quantified at ~1.3 mg/g algal dry weight. This article is protected by copyright. All rights reserved

Children as Bushfire Educators - 'Just be Calm, and Stuff Like That'

The limited evidence about disaster education programs for children indicates that they can increase disaster resilience, family based preparedness activities and reduce child fear and anxiety. However there are still gaps in the literature, including qualitative studies to provide insights into children’s experiences of disaster education programs, and follow up with children who are impacted by actual disasters. This paper reports on a pilot study in Victoria, Australia of a ‘Survive and Thrive’ bushfire education program delivered by the local Fire Brigade and incorporated into the school curricula for upper primary school children in Anglesea, a coastal town with very high bushfire risk. A mix of longitudinal qualitative methods captures the child experience and the program impacts including impact on child agency. The results show the children enjoyed the program and valued the life skills acquired. They demonstrated knowledge and skills gained in monitoring environmental risks and bushfire behaviour, and more nuanced understanding of the differential roles of adults and children in responding to a bushfire in different contexts. Faced with the reality of a nearby bushfire in the summer holidays, children demonstrated their capacity to apply their knowledge and to contribute to family response.

Green biologics: the algal chloroplast as a platform for making biopharmaceuticals

Most commercial production of recombinant pharmaceutical proteins involves the use of mammalian cell lines, E. coli or yeast as the expression host. However, recent work has demonstrated the potential of eukaryotic microalgae as platforms for light-driven synthesis of such proteins. Expression in the algal chloroplast is particularly attractive since this organelle contains a minimal genome suitable for rapid engineering using synthetic biology approaches; with transgenes precisely targeted to specific genomic loci and amenable to high-level, regulated and stable expression. Furthermore, proteins can be tightly contained and bio-encapsulated in the chloroplast allowing accumulation of proteins otherwise toxic to the host, and opening up possibilities for low-cost, oral delivery of biologics. In this commentary we illustrate the technology with recent examples of hormones, protein antibiotics and immunotoxins successfully produced in the algal chloroplast, and highlight possible future applications

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism

Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called "synthetic metabolism", may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering ("transplastomics") of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the "Design-Build-Test-Learn" (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants

Accelerating Chloroplast Engineering: A New System for Rapid Generation of Marker-Free Transplastomic Lines of Chlamydomonas reinhardtii

‘Marker-free’ strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter

ADA: an open-source software platform for plotting and analysis of data from laboratory photobioreactors

Algal biotechnology has received significant attention over the past two decades in fields ranging from biofuels to cosmeceuticals. However, the development of domesticated or genetically engineered microalgal strains for commercial applications depends on accurate and reliable growth data. To this end, several companies have developed lab-scale photobioreactors (PBRs) that enable precision control of conditions and automated growth recording. Whilst the transition from manual control of conditions and measurements to automated systems has allowed researchers to greatly improve the accuracy and scope of cultivation experiments, it has also presented novel challenges. The most pertinent of these being the analysis of the copious quantities of data produced. A standard PBR experiment can contain tens or even hundreds of thousands of data points, and often features outliers, noise, and a requirement for datasets to be calibrated with a standard curve or merged with replicates. Furthermore, complex analysis of multiple curves may be required in order to extract information such as the gradient or fit to a growth model. This can be laborious, time consuming and is not standardized between research groups. Proprietary software provided with most PBRs tends to lack these more advanced features and is typically unable to process data from other PBR manufacturers. To address these issues, we have developed the Algal Data Analyser (ADA), an open-source software platform providing the tools to rapidly plot and analyse microalgal data. ADA can simultaneously interpret datasets from three major PBR suppliers (Algenuity, Industrial Plankton, Photon Systems Instruments), and can also incorporate data from manual readings. Users can rapidly produce standardized, publication ready plots, and analyse multiple growth curves in parallel. Future iterations of ADA will include compatibility with datasets from other PBR suppliers as they become available, with the aim of making it a universal platform for all PBR data

Transgenic Microalgae Expressing Double-Stranded RNA as Potential Feed Supplements for Controlling White Spot Syndrome in Shrimp Aquaculture

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture

The experience of being a father of a son or daughter with an intellectual disability: older fathers’ perspectives

Background: As life expectancy for people with an intellectual disability increases, there is a growing cohort of older father carers. This study aimed to gain a more in‐depth understanding of older father carers’ experiences of parenting. Methods: Semi‐structured interviews were conducted with 7 older fathers (M = 63.9 years) and analysed using constructivist grounded theory. Results: Three conceptual categories were identified. “Wearing different hats: how fathers’ sense of identity had altered over the years. “Family comes first”: importance placed on the family unit. “Getting on in years”: the challenges faced by ageing fathers parenting their son/daughter. Conclusions: Fathers re‐evaluated their priorities and found a new identity in their parenting role, although they continued to see themselves as secondary carers. Fathers worried about the future as their health declined but drew strength from the benefits they had derived and the challenges that they had overcome to do their best for their son/daughter and their family