The Effect Of PotD、TldD And Glutamine Synthetase For Biofilm Formation In Escherichia Coli BL21(DE3) And Pseudomonas Putida F1

Biofilms are communities of bacteria embedded with extracellular matrix which consist of polysaccharide, protein and DNA when attached to biotic and abiotic surface. Majority of bacteria will inhabit in biofilm structure in nature. Bacteria of the matrix can be protected by the structure of biofilm. Exchanging nutrient and metabolites of bacteria in the biofilm matrix will be more effective and horizontal gene transfer in the biofilm will enhance the evolution. Serious advantages of ecology can be provided by the lifestyle of biofilm.The formation of biofilm can be affected by several factors, such as hydrological condition, surface, nutrient, light and temperature which are environmental factors. It can also be affected by the regulation of bacteria such as quorum sensing. Compared to planktonic growth phase differential gene expression will occur during the biofilm formation, which are related to adhesion processes, quorum sensing, metabolism, stress response and protein folding factor.The degradation of aromatic compounds has been widely studied biochemically or genetically. Through2-Dimensional gel electrophoresis (2-DGE) and Matrix assisted laser desorption/ionization-Time of flight (MALDI-TOF) analysis, the expression of mono cyclic aromatic compounds degradation related proteins were compared when bacteria Pseudomonas putida F1was grown on succinate, touene, ethylbenzene or the mixture of toluene and ethylbenzene as carbon and energy sources. Although the aromatic compounds were all metabolized using TOD pathway, the expression of some aromatic hydrocarbon degradation related proteins were different when the cells were grown on different carbon sources. Four groups of differentially expressed proteins have been identified, including metabolic proteins, transportation proteins, adaptation related proteins and other proteins. This study selects a clear genetic background of E. coli BL21(DE3), with a constant current simulation of natural fluid environment of the M9salt solution of low nutrient medium, glass wool adsorption media culture E. coli BL21(DE3), so that in the biofilm formed on the glass wool. E. coli BL21(DE3) and the process of biofilm formation observed through a microscope, and analysis of the biofilm formation, adhesion and aggregation stage, the micro-colony forming stages of biofilm maturation stage.Select a free state and biofilm state of the three protein expression differences, respectively putrescine/spermine transporter protein substrate binding subunit PotD, the protein metabolism TldD and glutamine synthetase by overexpression Save expression and gene knockout, the use of laser scanning confocal microscopy to study the impact of three genes of E. coli BL21(DE3) biofilm formation.Full-lengths sequence of potD, tldD, glnA gene in E.coli BL21(DE3) were searched from database. PCR amplified potD, tldD, glnA genes, then potD gene and the plasmid pET26b(+) were connected constructing excess potD gene over-expression vector pET26b(+)-fpotD; tldD and glnA genes, respectively, connected with plasmid pET28a(+) building the tldD gene and the glnA gene over-expression vector pET28a(+)-ftldD and pET28a(+)-fglnA. The antisense strand of the three genes into plasmid construct antisense nucleic acid vector pET26b (+)-ipotD, pET28a(+)-itldD and pET28a(+)-iglnA antisense vectors were generated after the target gene transcription anti-sense RNA, complementary binding with the target gene mRNA, interfering with the normal expression of the target gene, so as to achieve the reduction effect of the expression; Use of the Lambda-RED homologous recombination techniques for the purpose of gene knockout.The over-expression vectors pET26b(+)-fpotD, pET28a(+)-ftldD and pET28a(+)-fglnA were transformed into E. coli BL21(DE3) and were induced expressed, the expression of target proteins were verified by SDS-PAGE and Western blotting. Reduction of the expression vector pET26b (+)-ipotD, pET28a (+)-itldD and pET28a (+)-iglnA were transformed into E. coli BL21(DE3) at the same time. After antisense RNA were transcripted, Western blotting was used to verify the reduction effect of the expression. Constant current culture of each protein overexpression reductions expression strain deficient strain biofilm formation was observed the formation of biofilms Availability affect the analysis of the target gene on biofilm formation.The SOS repair DNA severely damage the cells in a critical state is induced by a DNA repair mode, the result of the repair is just able to maintain the integrity of the genome, to improve the rate of formation of the cells, but leave many errors, so the also known as error-prone the repair (the error-pronerepair), the cells have a higher mutation rate. Many bacteria, including E. coli, Salmonella, DNA damage or stalled DNA replication reacted SOS response. More than30genes involved in the SOS response, in which bacteria can increase DNA damage tolerance and DNA repair capacity. LexA protein is the main SOS response modifiers, until the DNA damage activates RecA protein with function as a transcriptional repressor. RecA-mediated and LexA self-digestion, thereby allowing the SOS gene expression. Therefore, the level of RecA expression can be used as to evaluate the the SOS response level of reliability indicators.Polyamines (putrescine, spermidine and spermine) are aliphatic cations exist in all organisms. The study proved to be the polyamine through non-covalent interactions in combination with proteins, nucleic acids, phospholipids, and other acidic substances, it is possible to influence the permeability of the cell membrane, gene expression and intracellular signal transduction and apoptosis. Because of their important regulatory functions, biosynthesis, degradation and absorption and excretion of polyamines is tightly regulated in order to maintain proper cell level. Recently, polyamines have been shown to mediate the production of RecA, with the the LexA control the SOS response regulator. The researchers found that groES, groEL, tldD, tldE and gyrA mutations can increase the strain the CcdB tolerated, but also in the the tld deletion mutant, the SOS response induced. Although TldD and TldE are proved to be associated with the secretion of antibiotics in containing pMccB17plasmid strain, they locate on chromosome, rather than plasmid, but no studies have shown, in addition to the outside TldE protein other proteins with TldD the have a similar effect. In order to study the relationship the PotD and TldD with the SOS system, we selected recA, sfiA, oxyR, groES, groEL and the gyrA to research the relationship with the SOS response genes,16srRNA was used as the internal control when RT-PCR were performed. Further analysis of the relationship between PotD TldD and SOS response were performed to analysis the relationship with the formation of biofilms.