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Nanoparticle Tracking Analysis (NTA) has been employed to measure the particle concentration and size distribution of magnetosomes extracted and purified from Magnetospirillum gryphiswaldense MSR-1, and then exposed to probe ultrasonication for various times, or 1% (w/v) sodium dodecyl sulphate (SDS) for 1 h. Particle concentration increased 3.7-fold over the first 15 min of ultrasonication (from 2 × 108 to >7.3 × 108 particles mL−1), but fell steeply to ∼3.6 × 108 particles mL−1 after 20 min. NTA of untreated magnetosome preparation confirmed a wide particle distribution dominated by larger species (D[1,0] = 312 nm; Dn50 = 261 nm; mode = 243 nm) with no particles in the size range of isolated single magnetosomes. After 5 min of ultrasonication the whole particle size distribution shifted to smaller size (D[1,0] = 133 nm; Dn50 = 99 nm; mode = 36 nm, corresponding to individual magnetosomes), but longer treatment times (15 and 20 min) reversed the previous transition; all characteristic numbers of the particle size distributions increased and very few small particles were detected. Side-by-side comparison of NTA and TEM sizing data revealed remarkable similarity at low ultrasonication times, with both showing single magnetosomes accounted for ∼30% population after 5 min. Exposure of magnetosomes to SDS resulted in a ∼3-fold increase in particle concentration to 5.8 × 108 particles mL−1, narrowing of the size distribution and gross elimination of particles below 60 nm. We conclude that NTA is a rapid cost-effective technique for measuring particle concentration, size distribution and aggregation state of magnetosomes in solution.

The over-expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition or activation of secondary metabolism pathways. Moreover, the expression of other plasmid-encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth kinetics.In this work, we have developed a second-generation system suitable for Escherichia coli expression with an antibiotic-free plasmid maintenance mechanism based on a glycine auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression was minimized by tuning the expression levels of the repressor protein (LacI) and glyA using a library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. The engineered antibiotic-free expression system was applied to the l-fuculose phosphate aldolase (FucA) over-production, showing an increase in production up to 3.8-fold in terms of FucA yield (mgg-1DCW) and 4.5-fold in terms of FucA activity (AUg-1DCW) compared to previous expression. Moreover, acetic acid production was reduced to 50%, expressed as gAcgDCW-1.Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity. © 2015 Elsevier B.V., This work was supported by the Spanish MICINN, project number CTQ2011-28398-CO2-01 and the research group 2009SGR281 and by the Bioprocess Engineering and Applied Biocatalisys Group, department of Chemical Engineering of the Universitat Autonoma de Barcelona, Cerdanyola del Valles (Spain). M.P. acknowledges the Universitat Autònoma de Barcelona for the pre-doctoral fellowship. Appendix A