Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme.

Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling bacterial microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about -210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins

Evaluation of the parfait blanket concept for fast breeder reactors

"January 1974."Also issued as a Ph. D. thesis by the first author and supervised by the second and third author, MIT, Dept. of Nuclear Engineering, 1974Includes bibliographical references (pages 261-264)An evaluation of the neutronic, thermal-hydraulic, mechanical and economic characteristics of fast breeder reactor configurations containing an internal blanket has been performed. This design, called the parfait blanket concept, employs a layer of axial blanket fuel pellets at the core midplane in the fuel pins of the inner enrichment zone; otherwise, the design is the same as that of the conventional LMFBR's to which the parfait configuration was compared. Two significant advantages were identified for the parfait blanket concept relative to the conventional design. First, the parfait configuration has a 25% smaller peak fast flux which reduces wrapper tube dilation by 37% and fuel element elongation by 29%; and second, axial and radial flux flattening contribute to a 7. 6% reduction in the peak fuel burnup. Both characteristics significantly diminish the problems of fuel and metal swelling. Other advantages identified for a typical parfait design include: a 25% reduction in the burnup reactivity swing, which reduces control rod requirements; a 7% greater overpower operating margin; an increased breeding ratio, which offsets the disadvantage of a higher critical mass; and more favorable sodium voiding characteristics which counteract the disadvantage of an 8% smaller power Doppler coefficient. All other characteristics investigated were found to differ insignificantly or slightly favor the parfait design.U.S. Atomic Energy Commission contract AT(11-1)-225

Remarks upon the mass oscillation formulas

The standard formula for mass oscillations is often based upon the approximation $t \approx L$ and the hypotheses that neutrinos have been produced with a definite momentum $p$ or, alternatively, with definite energy $E$. This represents an inconsistent scenario and gives an unjustified reduction by a factor of two in the mass oscillation formulas. Such an ambiguity has been a matter of speculations and mistakes in discussing flavour oscillations. We present a series of results and show how the problem of the factor two in the oscillation length is not a consequence of gedanken experiments, i.e. oscillations in time. The common velocity scenario yields the maximum simplicity.Comment: 9 pages, AMS-Te

Selection of Suitable Reference Genes for RT-qPCR Analyses in Cyanobacteria

Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works

Progress report no. 4

Statement of responsibility on title-page reads: editors: M.J. Driscoll, D.D. Lanning, I. Kaplan, A.T. Supple ; contributors: A. Alvim, G.J. Brown, J.K. Chan, T.P. Choong, M.J. Driscoll, G. A. Ducat, I.A. Forbes, M.V. Gregory, S.Y. Ho, C.M. Hove, O. K. Kadiroglu, R.J. Kennerley, D.D. Lanning, J.L. Lazewatsky, L. Lederman, A.S. Leveckis, V.A. Miethe, P. A. Scheinert, A.M. Thompson, N.E. Todreas, C.P. Tzanos, and P.J. WoodIncludes bibliographical referencesProgress report; June 30, 1973U.S. Atomic Energy Commission contract: AT(11-1)225

Progress report no. 3

Statement of responsibility on title-page reads: editors: M.J. Driscoll, D.D. Lanning, I. Kaplan; contributors: S. T. Brewer, G.J. Brown, P. Delaquil, M.J. Driscoll, G.A. Ducat, I.A. Forbes, M. V. Gregory, S.Y. Ho, M.S. Kalra, C.S. Kang, L.T. Kim, D.D. Lanning, J.L. Lazewatsky, T.C. Leung, E.A. Mason, N.R. Ortiz, N.C. Rasmussen, I.C. Rickard, K.D. Roberson, A.T. Supple, A.M. Thompson, and C.P. TzanosIncludes bibliographical referencesProgress report ; June 30, 1972U.S. Atomic Energy Commission contracts: AT(11-1)306

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P(trc) resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P(A1lacO-1), induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density