Screening Low-temperature Sensitive Promoter By Using The Synthetic Promoter Library (SPL) Technology

Low-temperature sensitive promoter is a kind of special promoter.Transcriptional intensity in low-temperature is higher than in normal temperature.Expression of heterologous protein can increase protein solubility;Some of heat sensitive protein very suitable for expressing at low-temperature.In nature,quantity of low-temperature sensitive promoters is very few.In order to research on characteristic of this type of promoter and construct new type of low-temperature expression vector.In this topic,by using the synthetic promoter library(SPL) technology,we have screened used synthetic low-temperature sensitive promoters and studied on those promoters.We have constructed suitable screening method for low-temperature sensitive promoters.By using resistance gene kan and enhanced green fluorescent protein gene (EGFP) as reporter gene,we contrastively researched on advantages and disadvantages of this two kinds of reporter gene,so we decided to adopt enhanced green fluorescent protein gene (EGFP) as reporter gene in this topic. Simultaneously, double-layer agar technique for inducing was established to preliminary screen promoters.By analyzing and comparing the promoter sequence of the four Escherichia coli cold shock protein CspA,CspB,CspG and CspI,consensus sequence aaa,taa, ttgc, tg, ttaat keeped invariant,and other sequence randomized.Than inverse PCR was amplificated using pColdⅢ-EGFP as templet,at last we constructed the synthetic promoter library containing approximately 1500 recombinant transformants.106 effective promoters were obtained by preliminary screening,and 17 low-temperature sensitive promoters were gotten by secondary screening,and finally 7 different low-temperature sensitive promoters were acquired by sequencing.Strong promoter P129and weak promoter P3 are relatively superior than other five promoters, and this two were chosen to detect characteristics.During process of analysis, when low-temperature sensitive promoter PCspAwas used as control,we found P129 and P3 were similar to PCspA in various aspects.The optimum expression temperature is 15℃; Prolong expression time is 45 h;The optimum expression cell age is logarithmic early growth(OD600 0.4 or so).By using Bacteria laccase gene as D15 as a reporter gene,we further detailed expressive effect of P129and P3.The results showed that detection results of using Bacteria laccase gene D15 as a reporter gene(SDS-PAGE detection) and using the Enhanced Green Fluorescence Protein (EGFP) gene as a reporter gene(fluorescence intensity detection) are consistent.In addition, comparing with expression status of D15 at T7 promoter, we found low-temperature sensitive promoters would not form inclusion bodies, but when T7 promoter initiated protein expression, it will form serious inclusion bodies.This showed that low-temperature sensitive promoters possessed certain advantages when expressed some protein.