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Conservative Site-specific Recombination and Phase Variation02:53

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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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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
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Nuclear reprogramming is the process of switching gene expression of one cell type to that of another cell type, usually from a differentiated cell state to an undifferentiated cell state. Differentiation occurs during processes such as development and morphogenesis, tissue regeneration, and malignancy. Cells can also be artificially induced to reprogram their gene expression by techniques such as nuclear transfer, induced pluripotency, and cell fusion. Such techniques have many applications in...
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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Next generation synthetic memory via intercepting recombinase function.

Andrew E Short1, Dowan Kim1, Prasaad T Milner1

  • 1Georgia Institute of Technology, School of Chemical and Biomolecular Engineering, Atlanta, GA, USA.

Nature Communications
|August 29, 2023
PubMed
Summary
This summary is machine-generated.

We developed a new synthetic memory technology that uses post-translational regulation to control recombinase function, enabling faster and more versatile genetic memory in living systems.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Genetic engineering

Background:

  • Transcriptional Programming enables decision-making in living systems.
  • Existing synthetic memory technologies have limitations in speed and capacity.

Purpose of the Study:

  • To develop a novel synthetic memory technology by regulating recombinase function post-translationally.
  • To enhance the speed, capacity, and functionality of synthetic memory in biological systems.

Main Methods:

  • Repurposing Transcriptional Programming parts to intercept recombinase function post-translation.
  • Implementing site-specific deletion for loss-of-function and inversion for gain-of-function.
  • Engineering nested Boolean logic operations for complex memory functions.

Main Results:

  • Achieved programmable loss-of-function and gain-of-function.
  • Demonstrated synthetic memory operations with nested Boolean logic.
  • Expanded memory capacity over 5-fold for a single recombinase with parallel multi-site reconfiguration.
  • Achieved ~10-times faster recombination speeds compared to previous methods.

Conclusions:

  • Interception synthetic memory offers a significant advancement in speed and capacity.
  • Post-translational regulation is key to the enhanced performance of this next-generation memory technology.
  • This technology is complementary to Transcriptional Programming, paving the way for intelligent synthetic biological systems.