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Related Concept Videos

Combinatorial Gene Control02:33

Combinatorial Gene Control

Combinatorial gene control is the synergistic action of several transcriptional factors to regulate the expression of a single gene. The absence of one or more of these factors may lead to a significant difference in the level of gene expression or repression.
The expression of more than 30,000 genes is controlled by approximately 2000-3000 transcription factors. This is possible because a single transcription factor can recognize more than one regulatory sequence. The specificity in gene...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Crossing Over01:34

Crossing Over

Unlike mitosis, meiosis aims for genetic diversity in its creation of haploid gametes. Dividing germ cells first begin this process in prophase I, where each chromosome—replicated in S phase—is now composed of two sister chromatids (identical copies) joined centrally.
The homologous pairs of sister chromosomes—one from the maternal and one from the paternal genome—then begin to align alongside each other lengthwise, matching corresponding DNA positions in a process called synapsis.
In order to...
Crossing Over01:30

Crossing Over

Crossing over is the exchange of genetic information between homologous chromosomes during prophase I of meiosis I. Genetic recombination gives rise to allelic diversity in the newly formed daughter cells. In humans, crossing over produces genetically distinct haploid egg and sperm cells that undergo fertilization to produce unique offspring. Before cell division starts, the germ cell’s chromosome(s) undergo duplication in the S phase of the cell cycle. As the cells enter prophase I, duplicated...
Law of Independent Assortment02:03

Law of Independent Assortment

While Mendel’s Law of Segregation states that the two alleles for one gene are separated into different gametes, a different question of how different genes are inherited remains. For example, is the gene for tall plants inherited with the gene for green peas? Mendel asked this question by experimenting with a dihybrid cross; a cross in which both parents are homozygous for two distinct traits resulting in an F1 generation that are heterozygous for both traits.
Law of Independent Assortment02:03

Law of Independent Assortment

While Mendel’s Law of Segregation states that the two alleles for one gene are separated into different gametes, a different question of how different genes are inherited remains. For example, is the gene for tall plants inherited with the gene for green peas? Mendel asked this question by experimenting with a dihybrid cross; a cross in which both parents are homozygous for two distinct traits resulting in an F1 generation that are heterozygous for both traits.

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Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity
12:31

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Published on: May 1, 2018

Sequence-independent construction of ordered combinatorial libraries with predefined crossover points.

Laetitia Jézéquel1, Jacqueline Loeper, Denis Pompon

  • 1Centre de Génétique Moléculaire du Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.

Biotechniques
|November 15, 2008
PubMed
Summary
This summary is machine-generated.

A new method, SIGNAL, enables efficient shuffling of protein segments to create mosaic enzymes. This approach aids in studying enzyme structure-function relationships and optimizing enzyme performance.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Combinatorial libraries are crucial for understanding enzyme structure-function relationships and optimization.
  • Existing methods for generating mosaic enzymes can be limited by sequence similarity requirements.

Purpose of the Study:

  • To introduce a novel method, SIGNAL (sequence-independent generation of a chimera-ordered library), for creating mosaic enzymes.
  • To demonstrate the utility of SIGNAL for shuffling protein segments and generating ordered combinatorial libraries.
  • To facilitate the functional optimization of enzymes through modular protein engineering.

Main Methods:

  • The SIGNAL method utilizes PCR to extract specific amino acid segments from parental enzyme templates.
  • A biotin-avidin-based technique is employed to minimize contamination from parental DNA.
  • Fusion PCR is used to reassemble complementary mosaic DNA fragments, enabling sequence-independent recombination.
  • The method is demonstrated through the construction of mosaic CYP2B enzymes.

Main Results:

  • SIGNAL allows for the efficient and modular shuffling of predefined amino acid segments between proteins.
  • The method is independent of sequence similarities between the proteins being recombined.
  • The technique effectively reduces parental template contamination in the final enzyme library.
  • The construction of mosaic CYP2B enzymes showcases the modularity and applicability of the SIGNAL approach.

Conclusions:

  • SIGNAL provides a versatile and robotizable platform for generating ordered combinatorial enzyme libraries.
  • This method significantly advances the ability to model structure-function relationships and optimize enzyme performance.
  • The sequence-independent nature of SIGNAL broadens its applicability to a wide range of protein engineering tasks.