A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept

Abstract Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of engineered functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5 and IS150 at up to 38 chromosomal loci in Escherichia coli, in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. The fitness cost of multiple IS-knockdown, detectable in flask-and-shaker systems was readily outweighed by the less frequent inactivation of the transgene, as observed in green fluorescent protein (GFP)-overexpression experiments. In addition, global transcriptomics analysis revealed only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multiple E. coli strains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

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CRISPR-interference based modulation of mobile genetic elements in bacteria

10.1101/428029 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ákos Nyerges ◽

Balázs Bálint ◽

Judit Cseklye ◽

István Nagy ◽

Csaba Pál ◽

...

Keyword(s):

Complete Removal ◽

Mobile Genetic Elements ◽

Industrial Biotechnology ◽

Chromosome Engineering ◽

Crispr Interference ◽

Genetic Elements ◽

Promising Tool ◽

Single Plasmid ◽

Bacterial Chromosomes

ABSTRACTSpontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of advantageous functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5, and IS150at up to 38 chromosomal loci inEscherichia coli,in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets, increasing the half-life of exogenous protein expression. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. Global transcriptomics analysis revealed nevertheless only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multipleE. colistrains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

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Direct measurement of electron-diffraction-pattern intensities using an energy loss spectrometer

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155311 ◽

1989 ◽

Vol 47 ◽

pp. 668-669

Author(s):

A. G. Jackson ◽

M. Rowe

Keyword(s):

Thermal Effects ◽

Dynamic Range ◽

Scattering Amplitude ◽

Dynamical Theory ◽

Site Symmetry ◽

Proof Of Concept ◽

Parallel Acquisition ◽

Kinematic Approximation ◽

Speed Up ◽

Structure Calculations

Diffraction intensities from intermetallic compounds are, in the kinematic approximation, proportional to the scattering amplitude from the element doing the scattering. More detailed calculations have shown that site symmetry and occupation by various atom species also affects the intensity in a diffracted beam. [1] Hence, by measuring the intensities of beams, or their ratios, the occupancy can be estimated. Measurement of the intensity values also allows structure calculations to be made to determine the spatial distribution of the potentials doing the scattering. Thermal effects are also present as a background contribution. Inelastic effects such as loss or absorption/excitation complicate the intensity behavior, and dynamical theory is required to estimate the intensity value.The dynamic range of currents in diffracted beams can be 104or 105:1. Hence, detection of such information requires a means for collecting the intensity over a signal-to-noise range beyond that obtainable with a single film plate, which has a S/N of about 103:1. Although such a collection system is not available currently, a simple system consisting of instrumentation on an existing STEM can be used as a proof of concept which has a S/N of about 255:1, limited by the 8 bit pixel attributes used in the electronics. Use of 24 bit pixel attributes would easily allowthe desired noise range to be attained in the processing instrumentation. The S/N of the scintillator used by the photoelectron sensor is about 106 to 1, well beyond the S/N goal. The trade-off that must be made is the time for acquiring the signal, since the pattern can be obtained in seconds using film plates, compared to 10 to 20 minutes for a pattern to be acquired using the digital scan. Parallel acquisition would, of course, speed up this process immensely.

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Remote Echocardiography: Proof of Concept for Support of National Disasters, Combat and Humanitarian Mission

PsycEXTRA Dataset ◽

10.1037/e452292006-001 ◽

2002 ◽

Author(s):

Sheri Yvonne Nottestad Boyd ◽

Linda L. Huffer ◽

Terry D. Bauch ◽

James L. Furgerson

Keyword(s):

Proof Of Concept ◽

Humanitarian Mission

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Model, simulation and experiments for a Buoyancy Organic Rankine Cycle

The European Physical Journal Applied Physics ◽

10.1051/epjap/2020200113 ◽

2020 ◽

Vol 92 (1) ◽

pp. 10906

Author(s):

Jeroen Schoenmaker ◽

Pâmella Gonçalves Martins ◽

Guilherme Corsi Miranda da Silva ◽

Julio Carlos Teixeira

Keyword(s):

Model Simulation ◽

Organic Rankine Cycle ◽

Thermodynamic Cycle ◽

Rankine Cycle ◽

Test Body ◽

Working Fluid ◽

Proof Of Concept ◽

Energy Scenario ◽

New Type ◽

Fluid Reservoir

Organic Rankine Cycle (ORC) systems are increasingly gaining relevance in the renewable and sustainable energy scenario. Recently our research group published a manuscript identifying a new type of thermodynamic cycle entitled Buoyancy Organic Rankine Cycle (BORC) [J. Schoenmaker, J.F.Q. Rey, K.R. Pirota, Renew. Energy 36, 999 (2011)]. In this work we present two main contributions. First, we propose a refined thermodynamic model for BORC systems accounting for the specific heat of the working fluid. Considering the refined model, the efficiencies for Pentane and Dichloromethane at temperatures up to 100 °C were estimated to be 17.2%. Second, we show a proof of concept BORC system using a 3 m tall, 0.062 m diameter polycarbonate tube as a column-fluid reservoir. We used water as a column fluid. The thermal stability and uniformity throughout the tube has been carefully simulated and verified experimentally. After the thermal parameters of the water column have been fully characterized, we developed a test body to allow an adequate assessment of the BORC-system's efficiency. We obtained 0.84% efficiency for 43.8 °C working temperature. This corresponds to 35% of the Carnot efficiency calculated for the same temperature difference. Limitations of the model and the apparatus are put into perspective, pointing directions for further developments of BORC systems.

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Indocyanine green-augmented diode laser therapy of telangiectatic and reticular leg veins: a clinical proof-of-concept trial

Endoskopie heute ◽

10.1055/s-0032-1308757 ◽

2012 ◽

Vol 25 (01) ◽

Cited By ~ 1

Author(s):

A Klein ◽

G Shafirstein ◽

E Kohl ◽

W Bäumler ◽

M Landthaler ◽

...

Keyword(s):

Indocyanine Green ◽

Laser Therapy ◽

Diode Laser ◽

Proof Of Concept

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Evaluation einer als Proof-of-Concept entwickelten MRT-kompatiblen und röntgentransparenten Knochenbohrmaschine

RöFo - Fortschritte auf dem Gebiet der Röntgenstrahlen und der bildgebenden Verfahren ◽

10.1055/s-0032-1311173 ◽

2012 ◽

Vol 184 (S 01) ◽

Author(s):

FV Güttler ◽

K Winterwerber ◽

C Gross ◽

A Heinrich ◽

M de Bucourt ◽

...

Keyword(s):

Proof Of Concept

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Besser als Genexpressionstests?

Onkologische Welt ◽

10.1055/s-0038-1649303 ◽

2018 ◽

Vol 09 (02) ◽

pp. 65-66

Author(s):

Alexander Kretzschmar

Keyword(s):

New York ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Proof Of Concept ◽

Zirkulierende Tumorzellen

Zirkulierende Tumorzellen (Circulating Tumor Cells; CTC) sind Prädiktoren von Spät-rezidiven bei Frauen mit einem Hormonrezeptor-positiven (HR-positiv), HER2-negativen frühen Mammakarzinom. Dies ergab eine Proof-of-Concept-Studie, die von Dr. Joseph Sparano, New York, auf dem SABCS 2017 vorgestellt wurde. Spätrezidive machen etwa die Hälfte aller Östrogenrezeptor-positiven Rezidive aus. Genexpressionstests können zwar das Risiko eines Rezidivs insgesamt quantifizieren. Sparanos Arbeitsgruppe glaubt aber, dass CTC bessere Biomarker für Spätrezidive sind.

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