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Related Concept Videos

Genomic DNA in Prokaryotes00:46

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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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Prokaryotes are small unicellular organisms that include the domains — Archaea and Bacteria. Bacteria include many common microorganisms, such as Salmonella and E. coli, while the Archaea include extremophiles that live in harsh environments, such as volcanic springs.
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pUdOs: Concise Plasmids for Bacterial and Mammalian Cells.

France O Manigat1,2, Louise B Connell1,2, Brittany N Stewart1,2

  • 1Center for Chemical and Synthetic Biology, Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.

ACS Synthetic Biology
|January 18, 2024
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Summary

New plasmids (pUdOs) offer flexible cloning and expression in bacteria and mammalian cells. These versatile vectors can be customized for various applications, potentially replacing traditional plasmids.

Keywords:
Escherichia coliShigellamammalian cell expressionplasmidproteobacteriatype III secretion system

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Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Small plasmids are essential tools in molecular biology for gene cloning and expression.
  • Existing plasmid systems may lack flexibility or compatibility across diverse host organisms.
  • The development of novel, adaptable plasmid backbones is crucial for advancing genetic engineering.

Purpose of the Study:

  • To introduce and characterize a new series of small plasmids, termed plasmids from the Université d'Ottawa (pUdOs).
  • To demonstrate the versatility of pUdOs as a backbone for both bacterial and mammalian expression vectors.
  • To highlight the adaptability of pUdOs for applications such as reporter systems and gene expression studies.

Main Methods:

  • Construction of 28 unique pUdO plasmids featuring diverse origins of replication and selection markers.
  • Characterization of the promoterless multicloning site, including insulation from spurious promoters and suitability for Golden Gate cloning.
  • Modification of pUdOs into expression vectors by promoter insertion and testing in bacterial (Shigella) and mammalian systems.

Main Results:

  • pUdOs exhibit flexible use in Escherichia coli and related gram-negative bacteria.
  • The promoterless multicloning site supports conventional and Golden Gate cloning.
  • pUdO-based vectors demonstrate efficient expression and transfection in mammalian cells, comparable to established plasmids.
  • A pUdO-based reporter system for Type III Secretion System in Shigella was successfully developed.

Conclusions:

  • The pUdO plasmid series provides a versatile and adaptable platform for molecular cloning and gene expression.
  • pUdOs can be readily converted into functional expression vectors for both prokaryotic and eukaryotic systems.
  • These novel plasmids offer a promising alternative to traditional plasmids, potentially enhancing a wide range of biotechnological applications.