Top PDF Engineering Enzyme Systems by Recombination

Engineering Enzyme Systems by Recombination

Engineering Enzyme Systems by Recombination

We constructed individual NNK libraries for each of these sites, and expressed mutants in E. coli BL21(DE3) ΔIlvD. We screened 90 variants from each library, using a medium throughput assay involving colorimetric derivatization with 2,4-dinitrophenylhydrazine (DNPH). This DNPH assay has a limit of detection of ~30 µM based on comparison with standard curves. Based on EcYfaW protein purification yields (~10 mg/L), we estimate that the cell lysate used in this screen had ~ 0.2 µM enzyme. Over 2 h, the YfaW variants needed to perform ~150 turnovers of 2 R -DHIV to be detected by the DNPH screen. Wild-type EcYfaW catalyzes ~3 turnovers/sec on L - rhamnonate, so if any of the YfaW variants had a fraction of wild-type activity on 2R-DHIV then they should be detected. No variants were found with improved activity in these single-site libraries. Since the position 33 histidine and the position 59 arginine are predicted to make hydrogen bonds with the C4 and C5 hydroxyls of L-rhamnonate that are not present in 2 R -DHIV (Figure 4- 1), we speculated that they may be particularly important sites for substrate binding. We made two- site combinatorial libraries with NNK codons at both H33 and R59, and screened 2000 variants. No variants were found with improved activity in this library, however.
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Efficient homologous recombination mediated genome engineering in zebrafish using TALE nucleases

Efficient homologous recombination mediated genome engineering in zebrafish using TALE nucleases

Fig. 1. In vivo recombination analysis. (A) Overview of HR detection system by co-injection of TALEN RNAs and a GFP-inserted targeting construct in zebrafish embryos. (B) TALEN target in the sox2 locus and a sox2-targeting construct. Black, gray and blue blocks represent ORF, 5 ′ UTR and 3 ′ UTR, respectively (sox2 has no introns). G (0 position, marked in red) just before the stop codon is duplicated in the targeting construct locus and the 2a-sfGFP fragment is inserted in between the Gs. (C) sox2 TALEN RNAs were injected without any donor DNA and its mutagenic activity was measured by PCR and restriction enzyme analysis. Each lane represents an NdeI-digested amplicon encompassing sox2 TALEN target sequence from genomic DNA isolated from five embryos. The NdeI-digested wild-type PCR products are 78 and 75 bp. (D-F) Lateral (D) and anterior-dorsal (E,F) views of 2 dpf wild-type embryo. Box marks dorsal diencephalon region. (F) Detection of sox2 transcripts by whole-mount in situ hybridization. Scale bar: 200 μ m. (G,H) Confocal microscope images of the brain region that is similar to that shown in the boxed areas in D-F. (G) An embryo injected with a circular form of a sox2 reporter targeting construct. (H) An embryo co-injected with circular form of the construct and sox2 TALEN RNAs. Groups of cells expressing sfGFP in the diencephalon region are indicated by arrows. Scale bar: 20 μ m.
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A Review on Engineering of Organophosphorus Hydrolase (OPH) Enzyme

A Review on Engineering of Organophosphorus Hydrolase (OPH) Enzyme

OPH is able to hydrolyze P-O phosphodiester bond in pa- raoxon, P-N phosphonofloride bond in DFP, sarin, and soman and P-S phosphorothioate bond in VX and P-CN phosphoroamidocyanide in tabun. Degradation rate of sub- strate of OPH, paraoxon is 10 8 M -1 sec -1 that is near the rate of diffusion (10 10 M -1 sec -1 ). However, hydrolyzing rate of P-S and P-F bonds are significantly low. In active site of the enzyme, a Lys-169 and a His-254 take part in hydro- lyzing reaction. Two metal divalent ions that are Zn in natural are bond to each other with the help of one hy- droxyl molecule that comprise nucleophile part. P=O bond in paraoxon substrate is activated by a tied co ordinance bond with Znβ 2+ and makes a weak complex with binding hydroxyl ion. Binding hydroxyl reaction developed by transmitting proton to a Lys so that nucleophile hydroxyl attack at phosphoric centre starts when the leaving group leaves. One water molecule enters the weak bond site of binuclear then the proton of water molecule is taken by His254 and is released in high pH3. OPH enzyme is clone and expression in many differ ent systems such as Escherichia coli, Drosophila melanogaster, pichia pasto- ris, Streptomyces lividans, insect cells Serratia, Arthrobac- ter, Enterobacter, Burkholderia, Flavobacterium and Pseudomonas diminuta 3.
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Systems Engineering for Software Engineers

Systems Engineering for Software Engineers

Before going on to discuss how we achieve this, we need to introduce our overall course structure and discuss, more specifically, how this has changed to accommodate systems engineering courses. At Lancaster, departments have a great deal of flexibility in designing course structures ranging from a very flat structure with many options to a hierarchical model where students have very little choice. The computer science course is an example of this latter type of structure where the majority of topics are core topics and must be taken by all students. Our computer science course is structured around three themes with practical project work cutting across and integrating these themes (Figure 1). Students must take 7 courses from each theme plus three further options.
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Systems Engineering for Software Engineers

Systems Engineering for Software Engineers

This is the first course that students encounter in the systems and software engineering stream where we don’t simply concentrate on software issues. Students taking this course have already taken courses in program and data structure design, introductory software engineering and software design. In introducing the course, we thought carefully about what we were trying to achieve and came to the conclusion that the fundamental goal of the course was what Checkland (Checkland 1981) calls systems thinking. That is, we wanted to expand the horizons of students so that they thought of problem-solving in systems and not just in software terms. When faced with a problem situation, we wanted them to be able to think critically about the problem as well as the possible solutions to the problem.
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Systems Engineering Essentials (in Aerospace)

Systems Engineering Essentials (in Aerospace)

NASA Intercenter Systems Analysis Team A Systems Engineering Conceptualization Source: NASA Langley Systems Engineering Handbook Concurrent Engineering = SE INTEGRATED PRODUCT TEAM INTEG[r]

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Engineering and technologies Umbilical Systems

Engineering and technologies Umbilical Systems

In the Offshore business segment Technip performs engineering, procurement, construction, installation, commissioning and the refurbishment/upgrading of offshore facilities for the oil & gas industry. Technip provides these services for fixed platforms in shallow water with conventional sub-structures and self installing platforms such as the TPG 500 and for deepwater facilities including Spar, semi-submersible, TLP, FPSO and FLNG units. Technip is a world leader in floatover installation of topsides and its R&D effort is focused on technology transfer for local content and new frontier areas such as ultra-deepwater and the Arctic.
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AC : WHAT IS SYSTEMS ENGINEERING?

AC : WHAT IS SYSTEMS ENGINEERING?

5. Information technology: A new discipline has emerged in the business schools and computer science departments that goes by the name information systems (which is often a minor in computer science departments) or management of information systems (usually in business schools). This definition of systems engineering tends to focus heavily on the interface of computers with the business world. 14 While this has emerged as a discipline in its own right, it appears that this definition is also related to the INCOSE definition. Students in this discipline are expected to become experts in database management, computer security, and transaction processing. In this respect, this definition significantly deviates from what is understood as systems engineering in industrial and electrical engineering departments. However, from the perspective of potential employers, this definition seems to be widely used on jobsites.
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The!Art!and!Science of!systems!engineering *

The!Art!and!Science of!systems!engineering *

This!work!culminates!years!of!experience!in!systems!engineering!and!focused! discussions!among!NASA!leadership,!systems!engineers,!and!systems!engineering! trainers! across! the! Agency.! One! consistent! theme! in! these! experiences! and! discussions! is! that! NASA! uses! many! definitions! and! descriptions! of! systems! engineering.! We! use! the! terms! and! job! titles! of! chief! engineer,! mission! systems! engineer,! systems! engineering! and! integration! manager,! system! architect,! vehicle! integration,! and! so! on! for! various! pieces! of! the! complete! systems! engineering! function.!We!need!to!agree!on!a!common!understanding!of!systems!engineering.!In! addition,!no!matter!how!we!divide!the!roles!and!responsibilities!among!people,!we! must! ensure!that!those!roles! and!responsibilities!are! clear!and!executed!as!a!functional! whole.! Our! objectives!are! to! provide! a!clear!definition! of!systems! engineering,! describe! the! highly"effective! behavioral! characteristics! of! our! best! systems! engineers!and!make!explicit!the!expectations!of!systems!engineers!at!NASA.
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Introduction to Process Systems Engineering

Introduction to Process Systems Engineering

Definition of Process Systems Engineering Holistic way PSE Design Control Operate Chemical   process   systems... Definition of Process Systems Engineering PSE Computer   science Chemica[r]

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HANDBOOK OF INTERCOM SYSTEMS ENGINEERING

HANDBOOK OF INTERCOM SYSTEMS ENGINEERING

A diagram to show the basic concepts of a device or system. Often the block diagram has system parameters such as transfer functions, gain, loss, level, DC voltage, inputs, outputs, and so on. I.) Block Diagrams are used on the Product Data Sheet to clarify the functions, show performance capabilities, and to show the input, output, control, and interconnection points. This diagram also defines and clarifies the specifications called out on the data sheet. II.) The diagrams called Block Diagrams are often, in fact, “Single Line Diagrams”. Single Line Diagrams are similar to the Block Diagrams but show more detail such as number of conductors in a cable, connector designations, connector details such as male/female, Equipment Model Numbers, and Equipment Designation Names/Numbers. At RTS™ Systems, these single line diagrams are called “System Block Diagrams” and are used for several purposes: 1. Act as a check list against the customer requirements. 2. Demonstrate to the customer the meeting of the customer’s requirements. 3. Are used to develop the equipment list (Lists the Quantities and Models numbers of equipment required to make the system). 4. Provide the information necessary to perform a system test. 5. Provide information to estimate wire and cable requirements. 6. Provide information to aid installation at the customer site. (Wiring Diagrams and Wire List can be generated from this information). 7. Graphically give a measure of the size and complexity of a given
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Systems Engineering Management Plan

Systems Engineering Management Plan

3. Systems engineering is an interdisciplinary approach and means to enable the realization of successful systems. It focuses on defining customer needs and required functionality early in the development cycle, documenting requirements, and then proceeding with design synthesis and system validation while considering the complete problem: operations, cost and schedule, performance, training and support, test, manufacturing, and disposal. SE considers both the business and the technical needs of all customers with the goal of providing a quality product that meets the user needs.
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Measuring systems engineering and project success : an evaluation of systems engineering practices and project duccess in a Dutch civil engineering contracting firm

Measuring systems engineering and project success : an evaluation of systems engineering practices and project duccess in a Dutch civil engineering contracting firm

The results do however confirm that the extent of SE application is different in each project. Although, all projects score an average SE score within a range of approximately 10% because the maximum score was 66% and the lowest score was 56%. The results suggest that SE elements are in some projects not applied while in other projects the same SE elements were applied quite substantially. This means Reef Infra does not apply a comprehensive and standardized SE method in their civil engineering projects. There are multiple reasons for the extent of SE application. First, unclear and out of date SE procedures affect the extent of SE application. Second, the level of SE skill and knowledge affected the extent of SE application. Third, Reef Infra is highly dependent on the client for the extent of SE application. Establishing clear SE procedures, enhancing SE skill and knowledge and reducing client dependency could enable the contractor to properly assess the extent of SE necessary to realize project success. A contingency approach to SE in construction projects can prevent over- and underinvestment. Relating SE performance to project success, enables proper recognition of how much SE activity is enough. This aspect must be further explored to establish clear methods for recognition. The approach to SE must be corresponding with the characteristics of the project, otherwise SE does not provide value in the project. Although, the measurement tool developed in the research emphasizes the extent of SE process applied at the project level it does provide the contracting firm with the necessary information to target underperforming SE tasks. Overall, to ensure that the contractor is capable of approach SE as a flexible process and not to overemphasize on SE tools, it is important the contractor must establish clear SE procedures, enhance SE skill and knowledge and reduce client dependency.
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Systems Engineering Guide for Systems of Systems Version 1.0 August 2008

Systems Engineering Guide for Systems of Systems Version 1.0 August 2008

In the second area, as plans for SoS upgrades are developed and implemented, the SoS systems engineer needs to assess progress in defining, planning, implementing, integrating and testing the changes made to affect the upgrade. The SoS systems engineer conducts the assessment as part of Orchestrating Upgrades to SoS . This assessment includes technical assessment of the changes in the individual systems that will be planned and implemented under the auspices of the program management and systems engineers of the constituent systems. In defining upgrades, the maturity of technologies to be incorporated is particularly critical in an SoS environment. Indicators of maturity include metrics such as version stability. The SoS systems engineer needs insight into the system-level work, but ideally system-level work is planned, implemented, and assessed as part of the constituent system’s SE process. Whether a member of the SoS SE team participates in the system reviews or the systems engineer for the constituent systems provides updates to the SoS systems engineer, technical assessment is based on the resources available and the criticality of the changes to the SoS. Good SE practice requires the SoS systems engineer to follow a disciplined technical review process for the SoS as defined in its SEP. The SoS systems engineer is specifically interested in system implementation progress that affects the SoS functionality, performance, or schedule (this is akin to the importance of critical IMS synchronization points to SoS SE) because these issues could be a source of risks for the SoS. Assessment encompasses functionality in the systems and the interfaces between each system and the other systems in the SoS to implement the SoS thread, including data communications and data utilization.
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RENSSELAER POLYTECHNIC INSTITUTE School of Engineering. Industrial & Systems Engineering

RENSSELAER POLYTECHNIC INSTITUTE School of Engineering. Industrial & Systems Engineering

This course provides a capstone and professional experience. Student teams work on independent projects in any field of industrial and management engineering approved by a faculty adviser. Typically, projects involve a manufacturing and service sector client who provides the student with an opportunity to gain an actual industrial experience. Memos, progress reports, and a final written and oral report are submitted to the project adviser and client. This is a communication-intensive course. Prerequisite: senior standing. Fall and spring terms annually. 3 credit hours

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Electro-Quasistatic Simulations in Bio-Systems Engineering and Medical Engineering

Electro-Quasistatic Simulations in Bio-Systems Engineering and Medical Engineering

There is a great number of applications in bio-systems engi- neering or medical engineering where slowly varying elec- tromagnetic fields play a key role. Some examples will be treated in this paper as there are: the electric activity of neurons on neurochips used as sensors in various applica- tion fields and the stimulating electric fields of implanted electrodes used for deep brain stimulation in patients with Morbus Parkinson and stimulation of the auditory nerves in deaf patients, respectively. Even though these processes happen in milliseconds they are quasistationary as the typ- ical dimensions, e.g. of the electrodes in the inner ear are small compared to the typical wavelength of the stimulation frequency. Quasistationary electromagnetic fields subdivide Correspondence to: U. van Rienen
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Engineering of homologous recombination hotspots with AU-rich sequences in brome mosaic virus.

Engineering of homologous recombination hotspots with AU-rich sequences in brome mosaic virus.

Construction of full-length recombinant RNA3 clones. Full-length cDNA clones of five different types of homologous recombinants (rec-R9, rec-AU1N, rec-AU1, rec-R91AU1, and rec-R91AU3) were constructed by replacing the 1,160-bp BamHI-EcoRI inserts of PN-R9 (Fig. 2A), PN-AU1N (Fig. 3B), PN- FIG. 1. Schematic representation of the 39 noncoding regions of wt BMV RNA1, N2-R91AU1 and DIC-R91AU1 RNA2, and PN-R91AU1 RNA3 constructs used for testing the effect of AU-rich sequences on homologous recombination. The PN-R91AU1 RNA3 molecule contains a ;1,250-nt-long, chimeric 39 noncoding region that consists of four elements (regions A to D). Region A contains wt RNA3 sequences located between positions 1 and 162 (counted from the 39 end [2]) and an upstream RNA1-derived region (positions 163 to 236 in wt RNA1). Region B contains sequences located between positions 7 and 200 in wt RNA3. Both regions A and B have a deletion (positions 81 to 100 in wt RNA3; shown by small rectangular boxes). Region C has a 765-nt sequence derived from cowpea chlorotic mottle bromovirus RNA3 (positions 24 to 788, counted from the 39 end [3]). Region D (shown as a dotted box) contains wt RNA3 sequences located between positions 220 and 297. PN-R91AU1 RNA3 also contains a 23-nt-long sequence (designated as R9; positions 196 to 219 in wt RNA2) and a 28-nt artificial AU-rich region (designated AU1). The locations of the restriction sites are indicated; oligonucleotide primers used for PCR are shown by short horizontal, numbered arrows. Arrow M depicts the position of the following mutagenesis oligodeoxynucleotides: 12, 65, 90, 91, 92, 95, 96, 128, 147, 148, 184, 192, and 203 (see Materials and Methods). N2-R91AU1 and DIC-R91AU1 RNA2s also contain R9 and the AU1 (enclosed within dashed lines). In both RNA2 mutants, the 39 noncoding sequence upstream of the R91AU1 region is derived from wt RNA2 (positions 220 to 293). N2-R91AU1 RNA2 has the downstream 39 noncoding sequence derived from wt RNA2 (positions 1 to 195), while the corresponding region in DIC-R91AU1 RNA2 is derived from wt RNA1 (positions 1 to 236). The h2 and h3 sequences are depicted by horizontal arrows.
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MODEL-BASED SYSTEMS ENGINEERING FOR CYBER-PHYSICAL SYSTEMS

MODEL-BASED SYSTEMS ENGINEERING FOR CYBER-PHYSICAL SYSTEMS

Detailed modeling Formal Models (Engineering, qualitative ) Reasoning Platform Rules checking D eci si on fe ed ba ck lo op Modeling (sub)domains DL (decidable) D ime nsi o[r]

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Writing a Systems Engineering Plan, or a Systems Engineering Management Plan? Think About Models and Simulations

Writing a Systems Engineering Plan, or a Systems Engineering Management Plan? Think About Models and Simulations

• Expectations: Programs whose system has external interfaces need to have dependencies (i.e., hierarchy) clearly defined. This should include interface control specifications, which should be confirmed early on and placed under strict configuration control. Compatibility with other interfacing systems and common architectures should be maintained throughout the

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THE DEVELOPMENT OF ENZYME SYSTEMS DURING EARLY INFANCY

THE DEVELOPMENT OF ENZYME SYSTEMS DURING EARLY INFANCY

peak in the hypothalamus at 10 days and in the cerebral cortex at 15 days of life, and then tapering off to adult levels. Histochem- ical studies indicated that the enzyme was more abund[r]

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