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Genome Size and the Evolution of New Genes03:21

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While every living organism has a genome of some kind (be it RNA, or DNA), there is considerable variation in the sizes of these blueprints. One major factor that impacts genome size is whether the organism is prokaryotic or eukaryotic. In prokaryotes, the genome contains little to no non-coding sequence, such that genes are tightly clustered in groups or operons sequentially along the chromosome. Conversely, the genes in eukaryotes are punctuated by long stretches of non-coding sequence.
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The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis...
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The organization of prokaryotic genes in their genome is notably different from that of eukaryotes. Prokaryotic genes are organized, such that the genes for proteins involved in the same biochemical process or function are located together in groups. This group of genes, along with their regulatory elements, are collectively known as an operon. The functional genes in an operon are transcribed together to give a single strand of mRNA known as polycistronic mRNA.
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The genome of most prokaryotic organisms consists of double-stranded DNA organized into one circular chromosome in a region of cytoplasm called the nucleoid. The chromosome is tightly wound, or supercoiled, for efficient storage. Prokaryotes also contain other circular pieces of DNA called plasmids. These plasmids are smaller than the chromosome and often carry genes that confer adaptive functions, such as antibiotic resistance.
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The evolution of new genes is critical for speciation. Exon recombination, also known as exon shuffling or domain shuffling, is an important means of new gene formation. It is observed across vertebrates, invertebrates, and in some plants such as potatoes and sunflowers. During exon recombination, exons from the same or different genes recombine and produce new exon-intron combinations, which might evolve into new genes. 
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Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
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Genetic Code Expansion in Pseudomonas putida KT2440.

Tianyu Gao1, Jiantao Guo2,3, Wei Niu4,5

  • 1Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 12, 2024
PubMed
Summary
This summary is machine-generated.

Researchers developed a method to genetically engineer Pseudomonas putida KT2440 by incorporating noncanonical amino acids (ncAAs) into proteins. This technique enhances the analysis and regulation of biosynthetic enzymes for biobased chemical production and bioremediation.

Keywords:
Amber suppressionGenetic code expansionNoncanonical amino acidsOrthogonal tRNA synthetase and tRNAPseudomonas putida KT2440

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Biotechnology

Background:

  • Pseudomonas putida KT2440 is a key microorganism for producing biobased chemicals and bioremediation.
  • Current methods for analyzing and engineering its biosynthetic enzymes are limited.
  • Genetic code expansion offers a way to enhance protein function and biological process control.

Purpose of the Study:

  • To establish a robust method for incorporating noncanonical amino acids (ncAAs) into proteins in P. putida KT2440.
  • To enable precise control and analysis of protein complexes and biosynthetic pathways.
  • To lay the groundwork for advanced genetic engineering of P. putida KT2440.

Main Methods:

  • Utilized genetic code expansion to incorporate two distinct ncAAs into proteins.
  • Employed orthogonal archaeal tRNA synthetase and tRNA pairs to respond to a UAG stop codon.
  • Demonstrated the method using superfolder green fluorescent protein (sfGFP) as a model protein of interest (POI).

Main Results:

  • Successfully incorporated two ncAAs into sfGFP in P. putida KT2440.
  • Established a reliable protocol for site-specific ncAA incorporation.
  • Validated the use of orthogonal archaeal tRNA synthetase/tRNA pairs for this purpose.

Conclusions:

  • The developed method provides a foundational tool for advanced genetic engineering of P. putida KT2440.
  • This technique facilitates the study and enhancement of biological functions in P. putida.
  • Enables precise manipulation of proteins for improved biobased chemical synthesis and bioremediation applications.