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Related Concept Videos

Cell Lines01:16

Cell Lines

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A cell line is a population of cells grown in vitro that can be subcultured over several generations. Normal cells cease to divide after a certain number of cell divisions, a process known as replicative senescence. This number, called the Hayflick limit, was conceptualized by Leonard Hayflick in 1961 when he observed that fetal cells grown in culture could only divide 40-60 times. This limit is due to the shortening of the telomeres during each round of cell division, preventing cell division...
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Lossless Lines01:23

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In electrical engineering, a lossless transmission line is characterized by a purely imaginary propagation constant and a resistive characteristic impedance. The ABCD parameters, which describe the relationship between the input and output voltages and currents, indicate an equivalent π circuit with an imaginary series impedance and a shunt admittance. This results in a transmission line that, when the product of the phase constant (beta) and the length of the line is less than pi, exhibits...
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Lossy Lines and Overvoltages01:22

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Transmission-line series resistance and shunt conductance cause three primary effects: attenuation, distortion, and power losses.
Attenuation
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Electric Field Lines01:25

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The three-dimensional representation of the electric field of a positive point charge requires tracing the electric field vectors, whose lengths decrease as the square of their distance from the charge and which point away from the charge at each point. This vector field is no doubt challenging to visualize. The visualization of electric fields becomes quickly intractable as the number of charges increases.
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Magnetic Field Lines01:19

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The representation of magnetic fields by magnetic field lines is very useful in visualizing the strength and direction of the magnetic field. Each of the magnetic field lines forms a closed loop. The field lines emerge from the north pole (N), loop around to the south pole (S), and continue through the bar magnet back to the north pole.
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Properties of Electric Field Lines01:25

Properties of Electric Field Lines

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The definition of electric field lines greatly eases the visualization of electric fields, a vector field, especially in the presence of many charges. The one-to-one correspondence between the electric field and the electric field lines necessitates that the field lines follow some rules.
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Generation of Stable Amber Suppression Cell Lines.

Simon J Elsässer1

  • 1Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. simon.elsasser@scilifelab.se.

Methods in Molecular Biology (Clifton, N.J.)
|February 7, 2018
PubMed
Summary

We developed a method to genetically incorporate noncanonical amino acids into proteins within living cells using amber suppression. This enables precise protein engineering for applications like cellular tagging and enzyme control.

Keywords:
Aminoacyl-tRNA-SynthetaseGenetic code expansionMammalian cellsNoncanonical amino acidsPiggyBac transposaseSite-specific protein labelingUnnatural amio acidstRNA

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Noncanonical amino acid mutagenesis allows protein tailoring within living cells.
  • The Methanococcus pyrrolysyl-tRNA synthetase/tRNACUA (PylRS/PylT) system has been used for incorporating diverse noncanonical amino acids in mammalian cells.
  • Previous applications include minimal genetically encoded fluorescence or affinity tagging, photo-control of enzymes, and genetically encoded posttranslational protein modifications.

Purpose of the Study:

  • To develop a general and efficient method for genomically integrating the PylRS/PylT amber suppression machinery.
  • To establish a protocol for generating stable amber suppression cell lines.
  • To achieve homogenous and highly efficient amber suppression in various cell lines.

Main Methods:

  • Utilized PiggyBac-mediated transposition for genomic integration of the PylRS/PylT system.
  • Developed a modular plasmid system for efficient delivery and expression.
  • Established a general protocol for creating stable cell lines capable of amber suppression.

Main Results:

  • Successfully integrated the PylRS/PylT amber suppression machinery into the genome.
  • Demonstrated a general protocol for generating stable amber suppression cell lines.
  • Achieved homogenous and highly efficient amber suppression across a range of mammalian cell lines.

Conclusions:

  • Genomic integration of the PylRS/PylT system via PiggyBac transposition provides an efficient method for noncanonical amino acid incorporation.
  • The developed protocol enables the generation of stable cell lines for broad applications in protein engineering.
  • This approach facilitates homogenous and highly efficient amber suppression, advancing the field of synthetic biology.