G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4 Sticky ends from different EcoO109I sites may not be compatible.Įfficient cleavage requires at least two copies of the NgoMIV recognition sequence.Įfficient cleavage requires at least two copies of the NaeI recognition sequence. Sticky ends from different BlpI sites may not be compatible. Sticky ends from different BtgI sites may not be compatible. PaeR7I does not recognize the sequence CTCTCGAG. Thus, the methodology described here can be adapted to investigate the activity and regulation of any promoter in L. Subsequently, analysis of fluorescence exhibited by cells carrying a single copy reporter can be performed under selected experimental conditions by stringent sample preparation, optimized image acquisition, and processing of the digital data with the image analysis freeware ImageJ. monocytogenes and integrated into its chromosome by homologous recombination within the selected promoter region. monocytogenes, a suicide shuttle vector carrying the Px::egfp gene fusion is first constructed in Escherichia coli (as an intermediate host). To construct a single copy of an EGFP-based fluorescent reporter fused to a promoter of interest (Px) in L. Here we describe the development of a chromosomally integrated transcriptional reporter fusion in Listeria monocytogenes that allows real-time measurements of gene expression. Reporter gene fusions based on the enhanced green fluorescent protein (EGFP) are powerful experimental tools that allow real-time changes in gene expression to be monitored both in single cells and in populations.
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