Stationary-phase Promoters Screening In Escherichia.coli And Construction Of PSP Expression Vector

When an Escherichia coli culture changes from exponential growth to the stationaryphase, expression of growth-related genes level off, while a number of stationary-phase genesare turned on, observably, there are a lot of stationary-phase promoters promotestationary-phase genes expressing in Escherichia coli genome. If we can isolate somestationary-phase promoters and use these promoters for constructing expression vectors, thesekinds of expression vectors will have the ability of stationary-phase expressing gene, and theycan not promote the gene to expresse until the Escherichia coli enters the stationary phase ofgrowth. In this way, we can also omit measuring OD data and adding IPTG.to the culturewhen we use some expression vector, such as pET expression vectors.To analyze which genes are stationary-phase genes, we used microarray assay of thetranscriptome of JM109 and BL21 with DNA chips in three growth phases, and found out thetranscriptomes that were stationary-phase expression.In addition to analyze the strength and specificity of the stationary-phase promoters, weforecast 20 promoters of stationary-phase genes using ReulonDB. For the in vivo assay ofpromoter activity, about 500 bp DNA fragments upstream from the translation initiationcodon were isolated and inserted into a newly report vector-β-Galactosidase vector. Theactivity of test promoters were determined by measuring theβ-Galactosidase protein (ODdata). Analysis of the culture time-dependent variation of 20 test promoters indicated that 4 ofthe stationary-phase promoters are up regulated in the stationary-phase.To optimize the activity of stationary-phase promoters, we construct pSP vectors whichhave Amp~r and Tet~r, a terminater in the upstream to restrain background expression, H-N tagand EK recognised site for protein separation and purification, a MCS site with sevenrestriction sites and a strong terminater in its downstream. These pSP vectors were confirmedby expressing calpastatin protein using SDS-PAGE, and got consistent results asβ-Galactosidase.