Effects Of Plasmids Commonly Used In Laboratory On The Whole Cell Proteins Expression Profile Of E.coli MG1655

The plasmid is a covalently closed circular double-stranded DNA, widely exists in microorganisms. It is different from the genome DNA and independent of the genome DNA. It can self-replicate, its size ranging from several kbs to several hundred kbs. Plasmid plays an important role in genetic engineering and widely used in this area. Plasmid commonly used in laboratory usually contains elements such as plasmid replication promoter, resistant gene, multiple cloning sites. Replicon determines plasmid copy number in host bacteria, the resistance gene is a selection marker, multiple cloning sites contain a variety of restriction enzyme sites to facilitate the insertion of a foreign gene, which is conducive to cloning and expressing of genes. However, what kind of impact the plasmid had on host bacteria protein expression profile is not yet clear yet. Therefore, this experiment will use the laboratory commonly used plasmids as the subject, MG1655as the host bacterial to explore which plasmid vector will impact the host bacteria significantly.Since E. coli has a clear genetic background, short growth cycle, fast growth, good security, simple operation, it has been commonly used in industrial production and scientific research. With the development of proteomics, all proteins expressed in E. coli could be measured. Therefore, this study will using proteomics technology to analyze the effects of laboratory commonly used plasmids on changes of whole-cell protein expression profile of E. coli. The experiment with the selected plasmids include suicide plasmid pXL275, cloning vector pACYC184, pEASY-T3, pUC19, pAK, expression vector pPROEX HTB, the helper plasmid pRK2013. The choice of host strain was MG1655, which is a classic K-12series of Escherichia coli. Electrotransfer was used in suicide plasmid, cloning plasmid and expression vector. Conjugation transfer was used as a method to introduce plasmid pRK2013into MG1655, the targeted strains were obtained through the corresponding resistance screening.Two-dimensional electrophoresis of MG1655with imported plasmids showed that:pXL275, pUC19, pAK, pPROEX HTB, pRK2013, pACU184could not cause significant changes in whole-cell protein profiles of MG1655, but the plasmid pEASY-T3had led to significantly changes in MG1655expression profiles. A total of33proteins with significant differences were detected by biological mass spectrometry. The effects of pEASY-T3on the expression profile of MG1655were:(1) Expression absence:phosphopentomutase, encoded by gene deoB; glutamate decarboxylase A, encoded by gene gadA; malate synthase A, encoded by gene aceB; gamma-amino butyraldehyde dehydrogenase, encoded by gene/wr; dipeptide transporter, encoded by gene dppA; super-penetration induced periplasmic proteins, encoded by gene osmY; universal stress protein, encoded by gene uspG; stress response protein, encoded by gene hdeA; acid resistance protein, encoded by gene hdeB; conserved protein YgiW, encoded by ygi W.(2) Protein expression was significantly reduced:isocitrate lyase, encoded by gene aceA; glycerol kinase, encoded by gene glpK; tagatose1,6-diphosphate aldolase, encoded by gene gatZ; dihydro lipoic acid turn succinyl enzyme, encoded by gene sucB; aldehyde dehydrogenase B, encoded by gene aldB; enoyl-acyl carrier protein reductase, encoded by gene fabI; glyoxal enzyme, encoded by gene hchA; iron binding and storage protein, encoded by gene dps.(3) Protein expression was significantly increased:β-galactosidase, encoded by gene lacZ (encoded by gene on plasmid vector); Beta lactamase, encoded by gene ampC (encoded by gene on plasmid vector).According to the differences of plasmid maps between pUC19and pEASY-T3. fl fragment was cloned and connected to vector pUC19, the bacteria MG1655/pUC19-fl was constructed, MG1655/pUC19was induced by IPTG, two-dimensional electrophoresis maps of the two strains showed that the expression of GadA was down-regulated, indicated that both LacZ and fl fragment had effects on GadA expression, while its specific mechanism should still to explore.Summary:Mass spectrometry results showed that plasmid pEASY-T3inhibited10genes expression in MG1655, resulting in a lack of protein expression.8proteins expression were down-regulated,2proteins were up-regulated. Among10genes that pEASY-T3directly inhibit, acid resistance related gene gadA is the focus of current research. It may had an impact on MG1655of its acidic resistance, affected the viability in acidic environments. It also demonstrated that plasmid pEASY-T3had a great interference on host cell protein expression, and may not be suitable for the function evaluation of a foreign gene as a vector.