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Construction of Yeast Two-hybrid System of the bHLH-PAS Domain of hPer1 cDNA

Wang Zheng-ron

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Abstract

Objective To construct yeast two-hybrid system of the bHLH-PAS domain of hPer1. Method The bHLH-PAS domain of hPer1 cDNA was amplified by PCR. The amplified fragment of hPer1 cDNA was ligased with the vector pGBKT7 to construct recombinant bait plasmid pGBKT7-hPer 1 bHLH-PAS. The cDNA library of human brain was constructed. The recombinant plasmid and the cDNA library were transfected into yeast strain AH109 by LiAc Method. The yeast two-hybrid system was screened by SD/-Ade/-His/-Leu/-Trp. Result Digested with endonuclease and sequenced, the recombinant plasmid of pGBKT7-hPer 1 bHLH-PAS was constructed correctly. The transformation rate was about 1.2×10 6/3 μg pGADT7-Rec. Conclusion Twenty-four positive colonies were selected by the yeast two-hybrid system. The positive colonies paved the way for further screening cDNA library and finding of unknown proteins interacting with the hPer1 protein.

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Objective To construct yeast two-hybrid system of the bHLH-PAS domain of hPer1. Method The bHLH-PAS domain of hPer1 cDNA was amplified by PCR. The amplified fragment of hPer1 cDNA was ligased with the vector pGBKT7 to construct recombinant bait plasmid pGBKT7-hPer 1 bHLH-PAS. The cDNA library of human brain was constructed. The recombinant plasmid and the cDNA library were transfected into yeast strain AH109 by LiAc Method. The yeast two-hybrid system was screened by SD/-Ade/-His/-Leu/-Trp. Result Digested with endonuclease and sequenced, the recombinant plasmid of pGBKT7-hPer 1 bHLH-PAS was constructed correctly. The transformation rate was about 1.2×10 6/3 μg pGADT7-Rec. Conclusion Twenty-four positive colonies were selected by the yeast two-hybrid system. The positive colonies paved the way for further screening cDNA library and finding of unknown proteins interacting with the hPer1 protein.

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Available abstract

Objective To construct yeast two-hybrid system of the bHLH-PAS domain of hPer1. Method The bHLH-PAS domain of hPer1 cDNA was amplified by PCR. The amplified fragment of hPer1 cDNA was ligased with the vector pGBKT7 to construct recombinant bait plasmid pGBKT7-hPer 1 bHLH-PAS. The cDNA library of human brain was constructed. The recombinant plasmid and the cDNA library were transfected into yeast strain AH109 by LiAc Method. The yeast two-hybrid system was screened by SD/-Ade/-His/-Leu/-Trp. Result Digested with endonuclease and sequenced, the recombinant plasmid of pGBKT7-hPer 1 bHLH-PAS was constructed correctly. The transformation rate was about 1.2×10 6/3 μg pGADT7-Rec. Conclusion Twenty-four positive colonies were selected by the yeast two-hybrid system. The positive colonies paved the way for further screening cDNA library and finding of unknown proteins interacting with the hPer1 protein.

Key concepts: Complementary DNA, Two-hybrid screening, Plasmid, cDNA library, Yeast, Recombinant DNA, Biology, Transformation (genetics)

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