Direct precise measurement of the stall torque of the flagellar motor in E. coli

The flagellar motor drives the rotation of flagellar filaments, propeling the swimming of flagellated bacteria. The maximum torque the motor generates, the stall torque, is a key characteristics of the motor function. Direct measurements of the stall torque carried out three decades ago suffered from large experimental uncertainties, and subsequently there were only indirect measurements. Here, we applied magnetic tweezer to directly measure the stall torque in E. coli. We precisely calibrated the torsional stiffness of the magnetic tweezer, and performed motor resurrection experiments at stall, accomplishing a precise determination of the stall torque per torque-generating unit (stator unit). From our measurements, each stator passes 2 protons per step, indicating a tight coupling between motor rotation and proton flux.

Download Full-text

Concentration determination of chromatin in unstained resin-embedded sections by means of Z-contrast in STEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100134521 ◽

1990 ◽

Vol 48 (2) ◽

pp. 186-187

Author(s):

M. Haider ◽

B. Bohrmann

Keyword(s):

Energy Loss ◽

Freeze Substitution ◽

Biological Macromolecules ◽

Local Concentration ◽

E Coli ◽

Concentration Determination ◽

Phosphorous Content ◽

Z Contrast ◽

Electron Energy Loss

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

Download Full-text

NOESY around the critical point Omega Tau c = 1.12. Precise determination of proton-proton distances and reorientation correlation times with two-field NOESY

Molecular Physics ◽

10.1080/002689700162621 ◽

2000 ◽

Vol 98 (4) ◽

pp. 201-210

Author(s):

Tao Zhang, Xi-An Mao, Matthias Baur, Ho

Keyword(s):

Critical Point ◽

Precise Determination ◽

Correlation Times ◽

Proton Proton

Download Full-text

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

ACADEMIA JOURNAL OF BIOLOGY ◽

10.15625/2615-9023/v40n4.11689 ◽

2018 ◽

Vol 40 (4) ◽

Author(s):

Dang Thi Ngoc Ha ◽

Le Thi Thu Hong ◽

Truong Nam Hai

Keyword(s):

Recombinant Antibody ◽

Blood Type ◽

Soluble Form ◽

Supernatant Fraction ◽

Blood Group Antigens ◽

Single Chain ◽

E Coli ◽

Recombinant Scfv ◽

Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

Download Full-text

Precise determination of 14 REEs in GSJ/AIST geochemical reference materials JCp-1 (coral) and JCt-1 (giant clam) using isotope dilution ICP-quadrupole mass spectrometry

GEOCHEMICAL JOURNAL ◽

10.2343/geochemj.2.0498 ◽

2018 ◽

Vol 52 (1) ◽

pp. 75-79 ◽

Cited By ~ 2

Author(s):

Tsuyoshi Tanaka ◽

Seung-Gu Lee ◽

Taehoon Kim ◽

Seunghee Han ◽

Hyo Min Lee ◽

...

Keyword(s):

Mass Spectrometry ◽

Reference Materials ◽

Isotope Dilution ◽

Precise Determination ◽

Giant Clam ◽

Quadrupole Mass ◽

Quadrupole Mass Spectrometry ◽

Geochemical Reference Materials

Download Full-text

PRECISE DETERMINATION OF THE IRRATIONAL PRE-FERRED INTERFACE ORIENTATION BY TEM

ACTA METALLURGICA SINICA ◽

10.3724/sp.j.1037.2010.00411 ◽

2010 ◽

Vol 46 (4) ◽

pp. 411-417 ◽

Cited By ~ 4

Author(s):

Yang MENG ◽

Lin GU ◽

Wenzheng ZHANG

Keyword(s):

Precise Determination ◽

Interface Orientation

Download Full-text

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207322666190412165316 ◽

2019 ◽

Vol 22 (5) ◽

pp. 346-354

Author(s):

Yan A. Ivanenkov ◽

Renat S. Yamidanov ◽

Ilya A. Osterman ◽

Petr V. Sergiev ◽

Vladimir A. Aladinskiy ◽

...

Keyword(s):

Antibacterial Activity ◽

Large Scale ◽

Underlying Mechanism ◽

Luciferase Assay ◽

Reporter System ◽

E Coli ◽

Starting Point ◽

High Concentrations ◽

Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

Download Full-text

Nauwkeurige methode voor de aanwasbepaling van het grondvlak met behulp van de Pressler-boor

Silva Gandavensis ◽

10.21825/sg.v12i0.1009 ◽

1968 ◽

Vol 12 ◽

Author(s):

R. Goossens

Keyword(s):

Basal Area ◽

Precise Determination ◽

Maximum Diameter ◽

Precise Method ◽

Maximum Radius ◽

The Core ◽

Two Parameters

A precise method for the determination of the increment of the basal area using the PressIer bore. Refering to previous research showing that the basal area of the corsica pine could be characterized by an ellips, we present in this paper a precise method for the determination of the increment of the basal area. In this method we determine the direction of the maximum diameter, we measure this diameter and we take a core in one of the points of tangency of the caliper with the measured tree. The determination of the diameter perpendicular to the maximum diameter finishes the work wich is to be done in the forest. From the classical measurements effectuated on the core and from the measured diameters we can then determine the form (V) and the excentricity (e). Substituting these two parameters in the formula 2 or 2', we can also calculate the error of a radius measured on the core with respect to the representative radius, This error with them allow us to correct the measured value of the minimum or the maximum radius and we will be able to do a precise determination of the increment.

Download Full-text