Engineering Bacillus megaterium for production of functional intracellular materials

Background: Over the last 10-15 years, a technology has been developed to engineer bacterial polyhydroxybutyrate (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. So far, beads have mainly been produced in recombinant Escherichia coli which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals. Results: In this study, we have engineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding β-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled/effected PHB production by B. megaterium PHA05. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40-50 mg IgG/g beads. As B. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads. Conclusions: This study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.fals

International Law: The Supreme Court Rules on Government Authorized Abduction

United States v. Alvarez-Machain, 112 S. Ct. 2188 (interim ed. 1992)

Fusion at deep subbarrier energies: potential inversion revisited

For a single potential barrier, the barrier penetrability can be inverted based on the WKB approximation to yield the barrier thickness. We apply this method to heavy-ion fusion reactions at energies well below the Coulomb barrier and directly determine the inter-nucleus potential between the colliding nuclei. To this end, we assume that fusion cross sections at deep subbarrier energies are governed by the lowest barrier in the barrier distribution. The inverted inter-nucleus potentials for the $^{16}$O +$^{144}$Sm and $^{16}$O +$^{208}$Pb reactions show that they are much thicker than phenomenological potentials. We discuss a consequence of such thick potential by fitting the inverted potentials with the Bass function.Comment: 8 pages, 5 figures. Uses aipxfm.sty. A talk given at the FUSION08: New Aspects of Heavy Ion Collisions Near the Coulomb Barrier, September 22-26, 2008, Chicago, US

Exact Stochastic Mean-Field dynamics

The exact evolution of a system coupled to a complex environment can be described by a stochastic mean-field evolution of the reduced system density. The formalism developed in Ref. [D.Lacroix, Phys. Rev. E77, 041126 (2008)] is illustrated in the Caldeira-Leggett model where a harmonic oscillator is coupled to a bath of harmonic oscillators. Similar exact reformulation could be used to extend mean-field transport theories in Many-body systems and incorporate two-body correlations beyond the mean-field one. The connection between open quantum system and closed many-body problem is discussed.Comment: Proceedings series of Proceedings of "FUSION08: New Aspects of Heavy Ion Collisions near the Coulomb Barrier", September 22-26, 2008, Chicago, US

Extraction of nucleus-nucleus potential and energy dissipation from dynamical mean-field theory

Nucleus-nucleus interaction potentials in heavy-ion fusion reactions are extracted from the microscopic time-dependent Hartree-Fock theory. When the center-of-mass energy is much higher than the Coulomb barrier energy, extracted potentials identify with the frozen density approximation. As the center-of-mass energy decreases to the Coulomb barrier energy, potentials become energy dependent. This dependence indicates dynamical reorganization of internal degrees of freedom and leads to a reduction of the "apparent" barrier. Including this effect leads to the Coulomb barrier energy very close to experimental one. Aspects of one-body energy dissipation extracted from the mean-field theory are discussed.Comment: 6 pages, 5 figures. Uses aipxfm.sty. A talk given at the FUSION08: New Aspects of Heavy Ion Collisions Near the Coulomb Barrier, September 22-26, 2008, Chicago, US