A DNA assembly toolkit to unlock the CRISPR/Cas9 potential for metabolic engineering

CRISPR/Cas9-based technologies are revolutionising the way we engineer microbial cells. One of the key advantages of CRISPR in strain design is that it enables chromosomal integration of marker-free DNA, eliminating laborious and often inefficient marker recovery procedures. Despite the benefits, assembling CRISPR/Cas9 editing systems is still not a straightforward process, which may prevent its use and applications. In this work, we have identified some of the main limitations of current Cas9 toolkits and designed improvements with the goal of making CRISPR technologies easier to access and implement. These include 1) A system to quickly switch between marker-free and marker-based integration constructs using both a Cre-expressing and standard Escherichia coli strains, 2) the ability to redirect multigene integration cassettes into alternative genomic loci via Golden Gate-based exchange of homology arms, 3) a rapid, simple in-vivo method to assembly guide RNA sequences via recombineering between Cas9-helper plasmids and single oligonucleotides. We combine these methodologies with well-established technologies into a comprehensive toolkit for efficient metabolic engineering using CRISPR/Cas9. As a proof of concept, we developed the YaliCraft toolkit for Yarrowia lipolytica, which is composed of a basic set of 147 plasmids and 7 modules with different purposes. We used the toolkit to generate and characterize a library of 137 promoters and to build a de novo strain synthetizing 373.8 mg/L homogentisic acid

Optimization of the centrifugal compressor return channel

The approach to optimizing the shape of the return channel blades for centrifugal compressor is described in this paper. A comparative analysis of two compressors flow paths is carried out. Values of the compressor relative polytrophic efficiency, the relative losses of the total pressure, as well as the distribution pattern of the streamlines in the return channel were compared during the analysis.В настоящей работе изложен подход к оптимизации формы лопаток обратного направляющего аппарата центробежного компрессора. Проведен сравнительный анализ двух вариантов проточной части компрессора. В ходе анализа сравнивались значения относительного политропного КПД компрессора, относительного коэффициента потерь полного давления, а также картины распределения линий тока в обратном направляющем аппарате

Testing of the flowing part design method in the centrifugal gas compressor

В работе кратко описывается разработанная методика проектирования проточной части центробежного компрессора, опирающаяся на подходы отечественных авторов. Реализовано автоматическое профилирование лопаточного аппарата компрессора и его меридиональных обводов на основании данных расчетов, что позволяет получать файлы с геометрий проточной части для дальнейшего использования в различных CAD/CAE пакетах. Проведена апробация программного кода на примере проектирования реального центробежного компрессора.The paper describes the developed methodology of design the centrifugal compressor flowing part, based on the approaches of Russian researchers. The blade profiling of compressor and its meridional contours are implemented on the basis of calculations, which allow to receive all geometry files of the flowing part for further automatical usage in some CAD/CAE-programs. Approbation of the in-house code is carried out on the example of the real centrifugal compressor design

Application of passive filters for improvement of electromagnetic compatibility of network and induction motor soft starters

В статье рассмотрена эффективность применения фильтрокомпенсирующего устройства с тиристорным преобразователем напряжения, используемым для организации плавного пуска асинхронного двигателя. Изложена методика расчета параметров фильтров, приводятся результаты моделирования, показывающие значительное снижение уровня высших гармоник при использовании фильтрокомпенсирующего устройства, сделан вывод о целесообразности установки ФКУ в тиристорных системах плавного пуска АД.The paper discusses using of passive filters with thyristor soft starter. Filter parameters calculation procedure is described. Simulation results show that using of passive filters greatly reduce higher harmonics levels

Исследование эколого-экономических показателей систем очистки газовых выбросов от диоксида серы

The paper presents the ecological and economical characteristics of different ways for purifying of waste gases from sulfurous anhydride (SO2).And proved that the least expenditures for purification of 1000 m3 have: lime and limestone, but also electron beam Methods. But, electron beam Method allow at the same time to carry out the purification of smoke gases from SO2 and NOх.Рассмотрены эколого-экономические показатели различных способов очистки газовых выбросов, содержащих сернистый ангидрит (ЅΟ2). Показано, что наименьшие затраты на очистку 1000 м3 имеют известковый и известняковый, а также электронно-лучевой методы. Однако электронно-лучевой метод позволяет одновременно производить очистку дымовых газов сразу от ЅΟ2 и ΝΟх

A study on L-threonine and L-serine uptake in Escherichia coli K-12

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptakesubsystem in E. coli cells

Digital receivers for low-frequency radio telescopes UTR-2, URAN, GURT

This paper describes digital radio astronomical receivers used for decameter and meter wavelength observations. This paper describes digital radio astronomical receivers used for decameter and meter wavelength observations. Since 1998, digital receivers performing on-the-fly dynamic spectrum calculations or waveform data recording without data loss have been used at the UTR-2 radio telescope, the URAN VLBI system, and the GURT new generation radio telescope. Here we detail these receivers developed for operation in the strong interference environment that prevails in the decameter wavelength range. Data collected with these receivers allowed us to discover numerous radio astronomical objects and phenomena at low frequencies, a summary of which is also presented.Comment: 24 pages, 15 figure

Experience in Optimization of Centrifugal Compressor Flow Path

In this paper, recommendations for carrying out multicriteria optimization of centrifugal compressor flow path are given. A step-by-step method of optimization has been developed.В настоящей работе приведены рекомендации к проведению многокритериальной оптимизации проточной части центробежного компрессора. Разработан пошаговый метод проведения оптимизационных работ

THE IMPACT OF THE COLLECTING CHAMBER CONSTRUCTION ON THE PRESSURE LOSSES IN CENTRIFUGAL COMPRESSOR OUTPUT DEVICE

The paper analyzes several variants of the centrifugal compressor collecting chamber embodiment for the output device pressure losses determination. For the most efficient construction the graph of the losses of the mass flow rate is deemed.В работе проанализированы несколько вариантов исполнения сборной камеры центробежного компрессора для оценки влияния конструкции на потери давления в выходном устройстве. Для наиболее эффективного варианта проведены расчеты зависимости потерь давления от массового расхода