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Abstract

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon . For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1–FPs that are functional in cell division and CetZ1–FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Funding
This study was supported by the:
  • Australian Research Council (Award FT160100010)
    • Principle Award Recipient: IainGeoffrey Duggin
  • Australian Research Council (Award DP160101076)
    • Principle Award Recipient: IainGeoffrey Duggin
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2024-05-24
2024-06-19
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