Please use this identifier to cite or link to this item: https://hdl.handle.net/20.500.13087/482
Title: Characterization and use of Tetrahymena thermophila artificial chromosome 2 (TtAC2) constructed by biomimetic of macronuclear rDNA minichromosome
Authors: Çalıseki, Mehmet
Dede, Ayça Fulya Üstüntanır
Arslanyolu, Muhittin
Keywords: Artificial chromosome
Telomere
rDNA locus
C3 origin
Recombinant
Chromosome stability and maintenance
Issue Date: 2021
Publisher: Elsevier Gmbh
Abstract: Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3 ' NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications.
URI: https://doi.org/10.1016/j.micres.2021.126764
https://hdl.handle.net/20.500.13087/482
ISSN: 0944-5013
1618-0623
Appears in Collections:PubMed İndeksli Yayınlar Koleksiyonu
Scopus İndeksli Yayınlar Koleksiyonu
WoS İndeksli Yayınlar Koleksiyonu

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