The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

Edible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during set-up and implementation of 'crop-protection' strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bio-available form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a Trp→Opal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the 'retro-fitting' of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish

Vitamin D Deficiency and Its Health Consequences in Africa

Africa is heterogeneous in latitude, geography, climate, food availability, religious and cultural practices, and skin pigmentation. It is expected, therefore, that prevalence of vitamin D deficiency varies widely, in line with influences on skin exposure to UVB sunshine. Furthermore, low calcium intakes and heavy burden of infectious disease common in many countries may increase vitamin D utilization and turnover. Studies of plasma 25OHD concentration indicate a spectrum from clinical deficiency to values at the high end of the physiological range; however, data are limited. Representative studies of status in different countries, using comparable analytical techniques, and of relationships between vitamin D status and risk of infectious and chronic diseases relevant to the African context are needed. Public health measures to secure vitamin D adequacy cannot encompass the whole continent and need to be developed locally

The chloroplast of Chlamydomonas reinhardtii as a platform for recombinant vaccine production

Infectious diseases represent an on-going problem adversely affecting the poultry industries and fish farming. Protein-base vaccines that are currently used for the prevention and spread of diseases have several drawbacks including high cost of recombinant production and delivery; and the needs to eliminate endotoxins when produced in bacterial platforms such E. coli. Therefore, there is an urgent need for effective, cheaper and safer vaccines. The microalga Chlamydomonas reinhardtii could be an ideal candidate for the production of recombinant protein such as vaccines. This is because: i) it has high growth rates; ii) it is generally regarded as safe (GRAS) to eat and therefore does not present a problem of endogenous toxin; iii) it has a genetically tractable chloroplast genome that allows a good expression of recombinant proteins that undergo correct folding to form an active product. Hence, in this research two C. reinhardtii transgenic chloroplast lines expressing the infectious bronchitis virus (IBV) muti-epitope genes (Spike Protein S540-564, Nulceocapsid Protein N67-83 and 120 residues of C terminus of the Nucleocapsid Protein) fused with the adjuvant, CTB and full length nervous necrosis virus (NNV) capsid gene were created. IBV causes respiratory disease in chickens. On the other hand, NNV causes cellular vacoulation and neuronal shape changes in fish. The IBV and NNV antigens were successfully detected anti-HA antibodies by western analysis. The lyophilised C. reinhardtii CTB-IBV that were fed to day 0 chicks, successfully triggered a mucosal and systemic immune response giving rise to antibodies against CTB, IBV and HA epitopes. The NNV fish vaccine trial is currently ongoing in Kasetart University, Thailand. Serum and mucus of NNV vaccinated fish will be analysed in the Purton Lab, UCL. The chloroplast of C. reinhardtii is an attractive organelle for the production of recombinant protein such as vaccines. However, the current level of recombinant protein production in the chloroplast of C. reinhardtii is normally less than 1% of total soluble protein. Therefore, in this study four C. reinhardtii mutant strains (UVM 2, 4, 10 and 11) were created through forward genetics. These mutants exhibit superior expression of recombinant genes in the chloroplast through forward genetics. These strains will be further analysed, so that they can be used to express recombinant genes such as peptide vaccine in the chloroplast of C. reinhardtii in the future

Rapid prototyping platform for Saccharomyces cerevisiae using computer-aided genetic design enabled by parallel software and workcell platform development

Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development

Immediate hypothermia reduces cardiac troponin I following hypoxic-ischemic encephalopathy in newborn pigs

Neonatal hypoxic-ischemic encephalopathy (HIE) is a clinically defined neurological condition following lack of oxygen and often associated with cardiac dysfunction in term infants. Therapeutic hypothermia after birth is neuroprotective in infants with HIE. However, it is not known whether hypothermia (HT) is also cardioprotective. Four newborn pigs were used in the pilot study and a further 18 newborn pigs (randomly assigned to 72h-normothermia (NT) or 24h-HT followed by 48h-NT) were subjected to global HIE insults. Serum cTnI was measured prior to and post the HIE insult. Blood pressure, inotropic support, blood gases and heart rate (HR) were recorded throughout. Cardiac pathology was assessed from histological sections. Cooling reduced serum cTnI levels significantly in HT pigs by 6h (NT, 1.36±0.67; HT 0.34±0.23 ng/ml, p=0.0009). After rewarming, from 24 to 30h post insult, HR and cTnI increased in the HT group; from HR[24h]=117±22 to HR[30h]=218±32 beats/minute (p=0.0002) and from cTnI[24h]=0.23±0.12 to cTnI[30h]=0.65±0.53ng/ml, (p=0.05). There were fewer ischemic lesions on cardiac examination (37%) in the HT group compared to the NT group (70%). Hypothermia (24h) pigs did not have the post-insult cTnI increase seen in NT treated pigs. There was a trend that HT improved cardiac pathology in this 3-day survival model