High Throughput Screening of Mutant PGK Promoter Based on Egfp
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Abstract:
The recombined vector pUC-PEBBK was constructed based on the pUC-19 as the base plasmid, which seclected PGK as the promoter and Egfp of the encodingenhanced green fluorescent protein as the reporter gene in this study. The recombinant cassette BATs-PGKp-Egfp-PGKt-KanMX-BATx was amplified by PCR,which was homologous recombination with Saccharomyces cerevisiae haploid AY15-α5 in order to construct the haploid recombinant α5-PEBBK. The expression of Egfp in Saccharomyces cerevisiae was detected using fluorescence microplateand fluorescence microplate. It was showed that the recombinant RFU of Egfp in the haploid recombinant α5-PEBBK was 523, which was 10.0 fold higher than the parental strain with RFU 53. It was concluded that the Egfp could be correctly expressed in Saccharomyces cerevisiae AY15-α5 under the control of PGK promoter. BSM1 was selected as the optimal medium for reducing the interference on fluorescence signal by the optimization of medium. The effects of inoculation quantity and time on Egfp fluorescence expression and microorganism growth were investigated. The optimal culture conditions were 40 μL of inoculation and 36 h of incubation time. The establishment of high efficient and sensitive high-throughput screening method provides a good foundation for the selection of subsequent series of expression intensity promoters.
