Droplet-based microfluidic screening and sorting of microalgal populations for strain engineering applications.

The application of microfluidic technologies to microalgal research is particularly appealing since these approaches allow the precise control of the extracellular environment and offer a high-throughput approach to studying dynamic cellular processes. To expand the portfolio of applications, here we present a droplet-based microfluidic method for analysis and screening of and , which can be integrated into a genetic transformation workflow. Following encapsulation of single cells in picolitre-sized droplets, fluorescence signals arising from each cell can be used to assess its phenotypic state.

A Simple Technology for Generating Marker-Free Chloroplast Transformants of the Green Alga Chlamydomonas reinhardtii.

The availability of routine methods for the genetic engineering of the chloroplast genome of Chlamydomonas reinhardtii is allowing researchers to explore the use of this microalga as a phototrophic cell platform for synthesis of high value recombinant proteins and metabolites. However, the established method for delivering transforming DNA into the algal chloroplast involves microparticle bombardment using an expensive "gene gun". Furthermore, selection of transformant lines most commonly involves the use of a bacterial antibiotic resistance gene.

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly.

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems.

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.

CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA.

Background: The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast.

New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii.

In recent years, there has been an increasing interest in the exploitation of microalgae in industrial biotechnology. Potentially, these phototrophic eukaryotes could be used for the low-cost synthesis of valuable recombinant products such as bioactive metabolites and therapeutic proteins. The algal chloroplast in particular represents an attractive target for such genetic engineering, both because it houses major metabolic pathways and because foreign genes can be targeted to specific loci within the chloroplast genome, resulting in high-level, stable expression.

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii.

There is a growing interest in the use of microalgae as low-cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein-coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool.

Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression.

Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene.

Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms.

Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host-bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time.

Haemophilus parainfluenzae has a limited core lipopolysaccharide repertoire with no phase variation.

Cell surface lipopolysaccharide (LPS) is a well characterized virulence determinant for the human pathogen Haemophilus influenzae, so an investigation of LPS in the less pathogenic Haemophilus parainfluenzae could yield important insights. Using a panel of 18 commensal H. parainfluenzae isolates we demonstrate that the set of genes for inner core LPS biosynthesis largely resembles that of H.

Structural studies of the lipopolysaccharide from Haemophilus parainfluenzae strain 20.

Haemophilus parainfluenzae is a Gram-negative bacterium that colonizes the upper respiratory tract of humans and is a part of normal flora. In this study, we investigated the lipopolysaccharide (LPS) expressed by H. parainfluenzae strain 20.

Telomeric expression sites are highly conserved in Trypanosoma brucei.

Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs) of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T.

Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum.

Background: African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion.

A novel ISWI is involved in VSG expression site downregulation in African trypanosomes.

African trypanosomes show monoallelic expression of one of about 20 telomeric variant surface glycoprotein (VSG) gene-expression sites (ESs) while multiplying in the mammalian bloodstream. We screened for genes involved in ES silencing using flow cytometry and RNA interference (RNAi). We show that a novel member of the ISWI family of SWI2/SNF2-related chromatin-remodelling proteins (TbISWI) is involved in ES downregulation in Trypanosoma brucei.