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DNA Replication02:40

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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
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The cell cycle is a series of events leading to DNA duplication followed by the division of cell content to form two daughter cells. The cell cycle progresses in four stages—the cell increases in size (gap 1 or G1-phase), duplicates its DNA (synthesis or S-phase), prepares to divide (gap 2 or G2-phase), and divides (mitosis or M-phase).
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Before a cell can divide, it must accurately replicate all of its chromosomes, including the DNA and its associated histone and non-histone proteins.  This process begins at numerous origins of replication during the S phase of the cell cycle in each of a cell’s chromosomes simultaneously. Certain nucleotides can act as origins of replication, but these sequences are not well defined - especially in complex, multi-cellular, eukaryotic species. The length of DNA that spans an origin...
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Tunable Expression Systems for Orthogonal DNA Replication.

Ziwei Zhong, Arjun Ravikumar, Chang C Liu

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    Summary
    This summary is machine-generated.

    Scientists enhanced gene evolution with new tools for the orthogonal DNA replication (OrthoRep) system. These expression parts allow higher gene expression, expanding applications for in vivo protein and pathway evolution.

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    Area of Science:

    • Synthetic biology
    • Molecular biology
    • Biotechnology

    Background:

    • The orthogonal DNA replication (OrthoRep) system enables rapid in vivo gene evolution.
    • Existing OrthoRep systems have limitations in gene expression strength due to specialized transcription components.

    Purpose of the Study:

    • To develop novel synthetic and evolved expression parts for the OrthoRep system.
    • To increase the expression levels of OrthoRep-encoded genes to match endogenous levels in Saccharomyces cerevisiae.

    Main Methods:

    • Engineering and testing of synthetic and evolved promoter mutations.
    • Incorporation of a genetically encoded poly(A) tail for expression tuning.
    • Evaluation of expression levels across different genes and OrthoRep systems.

    Main Results:

    • Developed new OrthoRep expression parts enabling tunable gene expression over a large range.
    • Achieved expression levels up to 43-fold higher than previously possible, reaching ~40% of the TDH3 promoter strength.
    • Demonstrated stable expression gains across passaging and consistent performance across multiple genes and OrthoRep systems.

    Conclusions:

    • The new expression parts significantly enhance the capabilities of the OrthoRep system.
    • Expanded applicability of OrthoRep for in vivo continuous evolution of proteins and pathways.
    • Provides a powerful tool for synthetic biology and metabolic engineering research.