Construction Of Protein Expression Monitoring System In Escherichia Coli K12 And Pseudomonas Aeruginosa PAO1

Genomics has been focused on by researchers during the 20th century. Then proteomics was being the main studying direction in the post-genomic era after the accomplishment of the "Human Genome Project". Various methods like 2-DE technique with Mass Spectrometry were usually used for isolating and identifying proteins. However, those techniques still couldn't meet the needs of proteome development. Therefore, exploring a high sensitive and high-throughput and large-scale research method is imperactive and being expected.In this study, Escherichia coli {E.coli) and Pseudomonas aeruginosa PAO1 were selected as host cells for constructing a protein expression monitoring system, respectively, using the reporter gene without its self-RBS. Since the reporter gene could share the RBS of the cloned gene, the transcriptional and translational levels of the cloned gene were depended on the intensity of luminescence so that the expression of the cloned gene could be determined by detecting the CPS. To verify the monitoring system constructed above and further to establish the fluorescence database for the corresponding protein of the whole-gemone of E.coli and PAO1 were going to be studied in the following research.The protein expression monitoring vectors were constructed in E.coli and been named pPROEX HTa-lux and pPROEX HTb-lux and pPROEX HTc-lux, repectively. pPROEX HTc-lux was the appreciate vector for the expression of luxABCDE in correct reading frame. The result of measuring the intensity of luminescence in E.coli containing pPROEX HTc-lux with IPTG showed the trc promoter could transcript the reporter gene luxABCDE, and the expression of the luxABCDE also could be induced by the regulating factor of trc promoter. Meanwhile, the region around the start codon of luxABCDE gene of pPROEX HTc-lux was sequenced to indicate that the sequencing region of pPROEX HTc-lux was matched with expected. Besides, the protein expression monitoring vectors were constructed in PAO1 which named pMS402-luxABCDE(a), pMS402-luxABCDE(b), pMS402- luxABCDE(c) as well.The protein expression monitoring systems established in E.coli and PAO1 in this study were new techniques which could achieve high-throughput and high sensitive and large-scale detection. This new method could be used for not only proteomics theory study, but also the practical fields like biomedical investigation.