Optimizing OTOP1 Gene Expression

OTOP1 gene sequences were obtained from the NCBI database for the following species: Gallus gallus (Gene ID: 422844), Xenopus tropicalis (Gene ID: 100495027), Chelonia mydas (Gene ID: 102932521), Dermochelys coriacea (Gene ID: 119854908), Papio anubis (Gene ID: 101001867), Mus musculus (Gene ID: 21906), and Homo sapiens (Gene ID: 133060). We optimized OTOP1 gene codons of the above species into codons for human expression systems. The OTOP1 gene sequences were synthesized and cloned into the pcDNA3.1 eukaryotic expression plasmid by Sangon Biotech (Shanghai). After screening positive clones with ampicillin resistance, sequencing was performed to obtain the pcDNA3.1 plasmid containing the correct OTOP1 sequence. All mOTOP1 point mutations were constructed using the Mut Express® II Fast Mutagenesis Kit V2 (Vazyme, C214). Point mutations of mOTOP1 were confirmed by sequencing.

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Tian L., Zhang H., Yang S., Luo A., Kamau P.M., Hu J., Luo L, & Lai R. (2023). Vertebrate OTOP1 is also an alkali-activated channel. Nature Communications, 14, 26.

Publication 2023

Mut Express® II Fast Mutagenesis Kit V2

Clones Codons Eukaryotic Gallus gallus Gene Human Mus musculus Mutagenesis Papio anubis Plasmid Point mutations Xenopus

Corresponding Organization :

Other organizations : Chinese Academy of Sciences, Kunming Institute of Zoology, Northeast Forestry University

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Variable analysis

independent variables

Species: Gallus gallus, Xenopus tropicalis, Chelonia mydas, Dermochelys coriacea, Papio anubis, Mus musculus, Homo sapiens

dependent variables

OTOP1 gene sequences optimized for human expression systems
OTOP1 gene sequences synthesized and cloned into the pcDNA3.1 eukaryotic expression plasmid
Screening of positive clones with ampicillin resistance
Sequencing to obtain the pcDNA3.1 plasmid with correct OTOP1 sequence
Construction of mOTOP1 point mutations using Mut Express® II Fast Mutagenesis Kit V2
Confirmation of mOTOP1 point mutations by sequencing

control variables

Use of the pcDNA3.1 eukaryotic expression plasmid
Use of the Mut Express® II Fast Mutagenesis Kit V2

controls

Positive control: Ampicillin resistance screening for selecting clones with the correct OTOP1 sequence
Negative control: Not specified

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