The outcome of this R&D effort will be the identification of hardware design approaches and efficient software algorithms that can be implemented as part of an ITS Upgrade and similar detector systems in other collider detectors. We have established a number of benchmark observables of heavy-flavor production at high and low-energy scales that became a strategic part for the future heavy-ion program at the LHC. Moreover, these observables connecting the precision of the single particle tracking and full jet reconstruction put stringent requirements on the performance of the new Si-detector developed for experiments at high collision rates. A major achievement was to establish a working environment for massive computing simulations using the High-Performance Computing clusters . The lightweight framework combines the conventional computing strategy with the possibility to leverage high capacity and parallelism within the HPC for the future simulation tasks. These developments, utilizing the multi-node and multi-core architecture will provide us with the necessary framework for the studies of the time-stamp based digitalization software for the purpose of future detectors. We have started the first tests of the novel setup. On the hardware side of the project, the main performance benchmarks of the principal elements of the readout system have been identified. The processing chip architecture will be defined within the first quarter of 2015 we will be able to devise the necessary hardware design approaches during the second year of the LDRD funding. Enabling more efficient flow of electrons across the boundary between living and human-made systems is critical for bio-energy technologies, including harvesting energy from wastewater and efficient synthesis of fuels from sunlight and CO2. Over the last 20 years, proteins that mediate electron transfer across this abiotic-biotic interface have been identified, purified,mobile vertical grow tables and structurally characterized in isolation. Yet, there remains a dearth of information about the in operando structure of the protein-material interface.

Our overall goal is to address these critical structure-function knowledge gaps so that we can redesign proteins for more efficient electron transfer to materials. The Mtr pathway of Shewanella oneidensis MR-1 is currently the best understood extracellular electron transfer pathway. It consists of a periplasmic decaheme cytochrome c , an outer membrane non-cyt c porin-like protein and an outer membrane decaheme cyt c . The outer membrane decaheme cyts c are the most unique and important components of this pathway since these are the proteins that transfer electrons to abiotic surfaces such as minerals or electrodes. However, there is little information about the protein-material interaction and there is no information on which amino acid residues of cyt c are recognized or associated with the material. This project seeks to uncover mechanisms of interaction between these cyt c and materials . Obtaining adequate amounts of outer membrane decaheme cyts c for structural studies is challenging because of intrinsic difficulty of expressing decaheme cyts c and their low solubility. This year we overcame these challenges to express and purify two variants of the decaheme cyt c, MtrF. First, we expressed a lipidated MtrF with a histidine-tag at its C-terminal. This protein was then purified in the presence of mild detergent using both affinity chromatography and ion exchange chromatography to greater than 90% purity with a net yield of ~1 mg protein/L culture. To eliminate the need for detergent, which will in turn facilitate structural characterization, we also expressed an MtrF variant lacking any lipid modification. To do so, we created a DNA construct containing the MtrB signal sequence, followed by the MtrF coding sequence and a Cterminal histidine-tag. This protein was secreted directly into the culture media, and we successfully purified it to greater than 90% purity at a yield of ~0.5 mg/L culture using a single affinity chromatography step.

We also characterized both the lipidated and non-lipidated MtrF to assess whether they are fully matured decaheme cyts c. UV-Vis spectroscopy of these proteins shows the characteristic peaks of c-type cytochromes at 408, 535, and 552 nm. Additionally, Electrospray Ionization Mass Spectrometry shows that the mass of these proteins is equal to the expected mass of decaheme MtrF. Thus, we conclude that these proteins are indeed fully matured MtrF. Lastly, we have established assays to measure the binding affinity of MtrF to inorganic materials. Specifically, we have shown that the fluorescence of MtrF is quenched upon exposure to certain materials. This measurement will allow us to determine the partition coefficient of MtrF for different materials, and thus greatly add to our understanding of the thermodynamics of proteinmaterial binding. Our most significant accomplishment has been the development of a flexible and inexpensive method for assaying the phenotypes of thousands of genes in parallel using transposon mutagenesis and DNA barcode sequencing. The key to the approach is the introduction of random DNA barcodes into the transposon. A mutant library for a given microbe is characterized a single time using the time-consuming and expensive TnSeq protocol. All subsequent assays to measure mutant fitness for thousands of genes in parallel only require the quantification of the DNA barcodes, a simple and inexpensive assay termed BarSeq. To date, we have applied RB-TnSeq to 21 diverse bacteria and generated over 3,000 whole genome mutant fitness profiles, representing ~9 million gene fitness measurements. We have identified phenotypes for over 20,000 bacterial genes including thousands of genes with no previous known function. We are using these data to predict gene function in diverse species using correlations in mutant fitness across hundreds of diverse growth conditions. In the current year of LDRD funding, we are working to extend the RB-TnSeq to additional, diverse microorganisms relevant to bio-fuel production, bioremediation, and nutrient cycling including archaea. In addition, we are working to increase the efficiency of transposon mutagenesis in diverse microorganisms by engineering new vector variants.

Lastly, we are developing computational tools to enable the microbiology community at large to mine our functional genomic datasets and to globally infer gene function across all sequenced microbial genomes using homology-based methods. There is a strategic imperative for investment in bio-manufacturing infrastructure at Berkeley Lab. In the 2013 State of the Union address, Barack Obama said: “I’m announcing the launch of three more of these manufacturing hubs, where businesses will partner with the Departments of Defense and Energy …. And I ask this Congress to help create a network of fifteen of these hubs and guarantee that the next revolution in manufacturing is Made in America.” While the complete specifications for these fifteen national manufacturing hubs have yet to be announced, given the Administration’s 2012 National Bio-economy Blueprint, it is very likely that there will be at least one biological manufacturing hub. This FY14 lab-wide strategic LDRD aims to place Berkeley Lab in a dominant position from which to lead a competitive effort that brings a bio-manufacturing hub to Northern California. While Berkeley Lab is uniquely positioned to leverage DOE investments in the JGI, NERSC, KBase, JBEI, and the ABPDU for bio-manufacturing competitive advantage,mobile vertical farm the capabilities and expertise at these facilities have yet to be integrated and successfully demonstrated as a ‘one stop-shop’ from target molecule identification to industry-ready microbial production strains. Operationally, this requires bio-manufacturing pipeline component standardization and interoperability; the ability to generate, QC, and track large numbers of DNA constructs; seamless integration of microbial strain construction with high-throughput functional assessment; and machine intelligence to learn from previous successes and failures to drive forward the next design iteration. This strategic LDRD aims integrate existing and develop new Berkeley Lab capabilities and expertise to create revolutionary bio-manufacturing infrastructure. This infrastructure will enable the rapid design, implementation, and assessment of target molecule production by iteratively uncovering and resolving critical biosynthesis bottlenecks. This LDRD aims to tackle a stress-test and a challenging biosynthesis demonstration project to drive a compelling success story narrative, while in parallel addressing key infrastructure gaps. Demonstrating the ability to go from target molecule to functional construct within a year for important and valuable targets will cement Berkeley Lab’s leadership in bio-manufacturing. Our major accomplishment in FY14 was to design and very nearly complete the DNA construction of a refactored actinorhodin antibiotic pathway . This is an impressive feat, as it constitutes a record-breaking refactored secondary metabolite pathway at 22 genes and 30 kbp in length. [In early FY15, we completed and validated the construction of the refactored actinorhodin pathway, transformed the construct into a modified Streptomyces coelicolor host lacking its native actinorhodin pathway, and detected the production of actinorhodin.] Sample preparation and analytical mass spectrometry methods were developed for actinorhodin and other secondary metabolites including violacein, our selected biosynthesis stress-test project target. The completion of the DNA construction of a 60,000-variant refactored violacein pathway combinatorial library is anticipated in the first half of FY15. Significant progress was also made in the development of machine learning algorithms for processing the violacein combinatorial library production data, and in the further development of DNA construction process tracking and assistance software.We succeeded in collecting “pseudo-SINBAD” data from our synchrotron beamline after careful re-alignment of the spindle. Collecting back-scattered reflections and extracting anomalous differences form them proved tractable, and indeed easier than expected.

The problems of non-isomorphism and reflecting crystal design proved to be significant, and we have new projects now investigating these two fronts. Non-isomorphism is a problem that must be overcome for any future multi-crystal macromolecular crystallography endeavor to move forward, and this fact is growing in appreciation in the XFEL and synchrotron communities alike. We discovered that ambient humidity can have a dramatic impact on isomorphism, and we are preparing a manuscript on this phenomenon, which suggested apparatus for controlling it. The design of a high-angle soft-X-ray monochromator also requires further development. This technology is critical for self-seeding in the soft and “tender” X-ray FELs currently under development, so we anticipate significant interest in it. What we learned is that the art of polishing and etching these crystals is absolutely key to preventing severe degradation of the beam emittance at each bounce, but existing manufacturing methods are optimized only for silicon and new etching protocols must be worked out for alternative materials. We also found that the current status of dynamical theory of reflection is severely lacking in the soft X-ray region, and we re-formulated this theory from first principles. We believe a highZ and large-cell material is optimal if crystals of sufficient quality can be prepared. Bismuth Telluride is a promising candidate. Nitrogen is an essential component of proteins and consequently a key element for life and cell development. Mineral N is often limited for plants, which consequently reduces plant growth and biomass yield. While this practice has been partly responsible for the ‘green revolution,’ it has come at high environmental and economic costs. In natural ecosystems, plants have developed strong relationships with microbes to cope with the low availability of essential nutrients such as N. For example, plant rhizospheres contain N2-fixing bacteria that are able to fix atmospheric N2 without the requirement of forming symbiotic association with a host-plant, however several of them depend on plant root exudates for carbon supply. Furthermore, endophytic bacteria colonizing root, stem and leaves of plants with N2-fixing function have been identified in several plants. The aim of this project is to optimize benefits from interactions between plants and free N2-fixing bacteria communities to provide adequate amounts of assimilable N to host-crops. This would reduce fertilizer consumption and carbon footprint of feed stock production, and greatly improve the sustainability of biomass production. Several additional N-fixing strains were isolated from plant isolates were phylo-typed based on 16S rDNA analyses. The genome sequences of several diazotrophic strains isolated were obtained through collaboration with JGI. Genomic comparison and extensive phenotypic characterizations were performed with a few of these strains. Most of the endophytes were capable of utilizing a broad range of carbon substrates. We analyzed the ability of the endophytic diazotrophs to fix atmospheric N2 under varying temperature in-vitro.Results indicated that N-fixation occurred optimally under optimal growth conditions. In addition, biochemical and genomic studies revealed that a bacterial strain could produce from a plant exuded compound the plant growth promoting plant hormone. This discovery led us to the engineering a plant to boost bacterial production of the plant hormone . Coupled with the ability to provide fixed N2 to the plants, this is an extremely desirable trait that can be utilized for sustainable agriculture of biofuel crops such as Switchgrass.