| This study contains two parts.1. Construction of the method on GC base specific mutagensisDirected evolution in vitro is a powerful tool for studying protein structure-function relations and improving protein property such as activity, thermostablity, solubility, expression and substrate specificity. In general, a successful directed evolution depends on two key procedures: construction of iversified target gene libraries and sensitive efficient screening methods. The former usually introduces mutation into target genes or recombine them in vitro, and clones modified DNA in order to generate new chacarter. In recent years, numerous different methods, including error-prone PCR, nucleotide Analogues method, mutator strain, chemical mutagenesis DNA shuffling, stagger extension process, random chimeragenesis on transient templates and so on, have been developed for providing a general reference for library construction. These methods have their own advantages and drawbacks which meet different requirements, however, none of them are specific to random mutagenesis of GC-rich gene.In this study, we developed a novel frequency-controlled mutagenesis strategy of GC base by combining sodium bisulfite modification with polymerase chain reaction (PCR). The target DNA molecule was firstly treated by sodium bisulfite, and some cytosines were partly deaminated to uracil becoming a heterozygotic DNA molecule containing uracil. Subsequently, PCR was performed to amplify the modified DNA, and uracils are base paired with adenines, resulting in transition of GC to AT.We investigated relationship between final mutation frequency and treatment time with sodium bisulfite, and found that mutation frequency increased by extending treatment time. The base mutations were introduced at the rate of 0.55%, 0.95%, 1.59%, 2.38% and 4.34% after treating for 1, 2, 4, 8 and 16 h, respectively. Correspondingly, amino acid mutation frequency reached 1.06% to 6.97%. This result reveal that desired and adequate mutation rate could be obtained simply through controlling treatment time. As expected, most of the mutations occurred at GC positions and transitions of GC to AT were predominant, which resulted in an obvious decrease in the GC content of the target gene. These characteristics is specific to GC-rich gene and helps to avoid the problem that GC-rich gene is difficultly expressed in prokaryotic host. The mutations appeared to be uniformly distributed in the target gene and didn’t form mutational hot spots, which was an important prerequisite for random mutagenesis.Using this method, we successfully constructed a diversified DNA library of bla gene encodingβ-lactamase and isolated seven variants which substrate specificity were altered. The results demonstrated that this method could yield a desired frequency of random mutation to DNA of interest, especially GC-rich genes, and provided a powerful tool for directed molecular evolution.2. Study of bacterium quorum sensing systemNumerous bacteria monitor their own population densities by secreting the small signal molecules, further regulate expression of specific genes and coordinate their behavior among family members in response to novel environmental challenges, this phenomenon is termed quorum sensing. Quorum sensing of Gram-negative bacterium are almost mediated by N-acyl homoserine lactone (AHL) family. During growth of bacteria, AHL molecule is released to environment and accumulated as increasing the cell concentration. Once AHL molecules reach a certain threshold concentrate, they bind with specific receptor proteins (luxR-type protein) produced by themselves to form complex. AHL-LuxR complex, which is a activating transcription factor, can recognize and bind specific DNA sequence {lux box) located in the promoter area and enhances expression of target genes.AHL molecules are involved in the regulation of different biological functions, including bioluminescence, plasmid conjugal transfer, induction of virulence genes, regulation of antibiotic production, biofilm formation and so on. Especially, pathogenecity of many pathogenic bacteria is regulated by quorum sensing system. So it is of great significant to further study on quorum sensing regulation mechanism.On the other hand, AHL molecules play important roles in pathogenecity of many bacteria, so a novel strategy for prevention of diseases is developed by limiting concentration AHL molecule. So isolation of AHL degrading bacterium and enzyme helps to construct of novel biological pest control engineering bacteria and transgenic plant.In this study, we investigated the ability producing and degrading AHL of some bacteria which were supplied by Marine Culture Collection of China and firstly found that Pseudaminobacter salicylatoxidans could produce AHL-type signal molecule and the production of signal molecule was increasing over time, to the hightest concentration in index growth and followed by stationary phase maintaining a superior concentration. Besides, we found seven strains which showed the ability of degrading AHL. They are Alcanivorax dieselolei、Rhodobacter sp.、Pseudoalteromonas byunsanensis、Halomonas sp.、Marinobacter hydrocarbonoclasticus、Alcanivorax venusti、Martelella mediterranea. Except Rhodobacter sp., others are all firstly found able to degrading AHL. We did a preliminary study about property of these seven strains, and found that Alcanivorax dieselolei、Rhodobacter sp. and Halomonas sp. have strong ability of degrading AHL, meanwhile these enzyme can be release into the extracellular. So thet are valuable to further study.By studying the quorum sensing system, we found the existing strains for AHL bioassay have their own drawbacks, especially when constructing the gene library of degrading AHL to screen. It is required very complex operation and expensive chemical reagents, so according to the principle of quorum sensing, we constructed a new biosensor strain using lux element (luxR gene and Plux promoter) derived from Vibrio fischeri and man gene encodingβ-mannanase from Bacillus subtilis. Besides, we established two ways including Congo red staining and DNS to detect AHL molecules by understanding the man property. Our study shows that the new biosensor strain has sensitive response ability to AHL molecule, easily assayable reportor trait and also can be used to detecting AHL. |
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