Engineering a Plasmid as a Reporter System for Quantifying Gene Expression in Escherichia Coli
Keywords:DNA cloning, restriction site, promoter, LacZ gene, β- galactosidase assay, RNA synthetic biology
Different gene reporter assays are used for quantifying various methods for synthetic control of gene expression. The activity of a reporter protein, fluorescence or chemical stain, is usually proportional to the level of mRNA produced in the cell and can be utilized for monitoring the activity of a promoter or enhancer. Such a reporter gene is the lacZ from Escherichia coli, encoding β- galactosidase. We cloned the PL promoter from phage lambda into the pRS414 plasmid of over 10kb to create a gene reporter assay. A KpnI restriction site was introduced into the plasmid used for inserting the promoter together with a ribosomal binding site for bacterial and archaeal mRNA and the initiation of transcription codon ATG in frame with the lacZ. B-galactosidase activity assays showed a correlation between the induced expression of the lacZ gene and the time, indicating the rightness of the construct, which can be used for lacZ reporter gene activity assays and synthetic control of gene expression in synthetic biology studies. The new plasmid named pRS414ge is very suitable for RNA synthetic biology experiments.
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LicenseCopyright (c) 2022 Proceedings of the Bulgarian Academy of Sciences
Copyright (c) 2022 Proceedings of the Bulgarian Academy of Sciences
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