E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

Prodigiosin, a red linear tripyrrole pigment and a member of the prodiginine family, is normally secreted by the human pathogen Serratia marcescens as a secondary metabolite. Studies on prodigiosin have received renewed attention as a result of reported immunosuppressive, antimicrobial and anticancer properties. High-level synthesis of prodigiosin and the bioengineering of strains to synthesise useful prodiginine derivatives have also been a subject of investigation. To exploit the potential use of prodigiosin as a clinical drug targeting bacteria or as a dye for textiles, high-level synthesis of prodigiosin is a prerequisite. This review presents an overview on the biosynthesis of prodigiosin from its natural host Serratia marcescens and through recombinant approaches as well as highlighting the beneficial properties of prodigiosin. We also discuss the prospect of adopting a synthetic biology approach for safe and cost-effective production of prodigiosin in a more industrially compliant surrogate host.

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Viruses such as influenza and Ebola are enveloped in lipid bilayers annexed from host cells and containing glycoproteins essential for the infection process. At the molecular level little is known about the assembly process in terms of physical interactions between the lipids and glycoproteins. In this paper we assemble HIV glycoproteins in lipid vesicles in order to examine envelope assembly, a process that is usually only executed under control of a host cell. Using atomic force microscopy it was possible to observe fusion of individual envelope like particles, and contrast this with the behaviour of lipid vesicles without envelope glycoproteins. It was found that the inclusion of glycoproteins caused the vesicles to distort and that the subsequent fusion "footprint" with a lipid bilayer was related to the envelopes' unique morphology. This non-spherical morphology suggests that the presence of a viral capsid may be essential for the stability of an enveloped virus. Interactions between trans-membrane gp41 and gp120, the spikes protruding from a virion, were examined using supported lipid bilayers. Interactions between the gp120 and membrane-located gp41 resulted in the assembly of unusual molecular wires, one molecule in height and with a zigzag arrangement of gp120 molecules. In this work we have shown that purely physical/chemical interactions have dramatic effects on glycoprotein/lipid assembly and should be considered in the development of virus based technologies such as virosomes.