Degradation Of Nitrobenzene Extraction And Characterization Of Bacteria Of Pseudomonas Xn-1 Cells Plasmid

The nitrobenzene degradative plasmid in Pseudomonas XN-1 is discussed in this paper, based on the characterization of nitrobenzene degradation by strain Pseudomonas XN-1, which attempts to do some prepared work for the research in molecule level of nitrobenzene degradation by this strain.The antibiotic resistance of this strain is detected, because it generally relates to the plasmid. The result shows that this strain has not antibiotic resistance to chloramphenicol and tetracycline, but ampicillin.The plasmid extraction from wild strains needs an appropriate method. First, lysis by alkali was used to extract plasmid from strain Pseudomonas XN-1. But the expected result had not been got, and a lot of cracked plasmid DNA fragments were got only.Then the strain cell is detected to determine weather there is any plasmid in the cell. The detection result analysed by gelose gel electrophoresis shows that the cell harbors a plasmid, and this plasmid is relatively large.So some methods suitable to large plasmid extraction, including lysis by SDS and a method from a literature, were used to try to extract the large plasmid from the strain cell. The lysis reactions in these two methods are gentle, so the large plasmid cannot be injured in the lysis process, opposite to lysis by alkali. It would be helpful to keep the integrality of the large plasmid during the extraction. The extraction results show that the clear and integral plasmid DNA bands were got using these two methods. And the gelose gel electrophoresis indicates that this large plasmid is about 22kb.To determine the plasmid function, the experiment is done to cure the plasmid from the cell, which will cause some change on the strain physiology. The result show that after curing the plasmid, the strain cell cannot grow on the selective medium containing nitrobenzene. This result indicates that the plasmid relates to nitrobenzene degradation. At the same time, some results indicate that this plasmid has nothing to do with the antibiotic resistance, because the antibiotic resistance of the strain remains after the plasmid curing.A few mutants were found on the plate containing nitrobenzene selective medium. Their colony appearances have some difference from the wild strain. Some evidences show that their growth characteristics on the selective medium are related to the plasmids harbored in their cells. A mutant probably harboring more plasmids can grow better on nitrobenzene selective medium than other strains. The plasmid harbored in this mutant is also a few kb smaller than others. So a probability is supposed that a certain plasmid DNA fragment deletion made the number of plasmid copy change, which affected the mutant growth on the selective medium.To set up the physical map of this plasmid, some restriction enzymes are used to cut the plasmid into several plasmid DNA fragments. But the results of restriction enzyme reaction are not satisfied. There may be two reasons which lead to these results: one is that the restriction enzymes are not suitable for this plasmid, that is to say there are no appropriate cut sites on the plasmid for these restriction enzymes; and the other is that the purification of the plasmid is not enough for restriction enzyme reaction, and some impurity affects the last results.