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The Research of Regulation Mechanism for Integron Capturing Gene Cassettes

Author: YangZeHua
Tutor: LvYuan;GuanMing;JiangXiaoFei;JiangXiaoHua
School: Fudan University
Course: Clinical Laboratory Science
Keywords: Integron Integrase Resistance Recombinant Lateral gene transfer
CLC: R346
Type: PhD thesis
Year: 2008
Downloads: 231
Quote: 0
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Bacterial resistance is very serious, especially the emergence of multi-drug-resistant strains is a huge challenge for clinical bacterial infectious diseases. The emergence of strains of antibiotic resistant single might be expected of the people, may be the result of genetic mutation caused resistance to multiple antibiotics, then at the same time in the same strains, it is difficult to explain the genetic mutation. Now studies have shown that the genetic material in bacteria and strains between the horizontal transmission of bacterial resistance an important means. Studied a group of healthy people from the community and at least one month is not taking antibiotics, the isolated E. coli integron frequency of occurrence of 15%. Jones et al's study indicates carry integrons strains than the strains carrying integron resistance to multiple antibiotics more prone. So far, has found more than 70 kinds of resistance gene cassette. These data suggest that the integration of sub play an important role in the horizontal transmission of resistance genes as well as the emergence of multi-resistant strains. Integron gain a survival advantage in some cases by capturing enabling the gene cassette, such as a drug resistance gene cassette, but may also adversely affect capture certain toxic to the host to produce the gene cassette, or excessive capture gene cassette cause some burden on the host bacteria breeding. Bacterial integron capture foreign gene cassette, must have a complete and fine adjustment mechanism, but this mechanism is still not clear. For the study of this mechanism will help to deepen the understanding of the integration of sub role in the horizontal spread of resistance genes, for the clinical development of effective drugs to block or slow down this process to provide useful help. Capture gene cassettes designated to take advantage of the integration of the sub-directional characteristics, the development of a recombinant tool to lay the foundation. For this reason, we will affect the integrated sub-on four aspects of capture gene cassette factors are host factor gene cassette itself, mainly the length of the gene cassette, external environmental factors, primarily the concentration of the antibiotics, the integration of the level of enzyme expression. The efficiency of 1. Integration of previously widely used in the determination of the new method for determination of integron capture efficiency of gene cassette recombination reaction catalyzed by the enzyme to using bonding experiments using phenotypic screening approach. This method is cumbersome, time-consuming, relative precision low integron not easy to capture in-depth study of the mechanism of gene cassette. Therefore, we have established a fluorescence-based quantitative PCR techniques, from the molecular level to determine the method of integration frequency. First, we use the pUC19 as vector high expression of integrin enzyme recombinant plasmid pUCINT of pACYC184 vector position respectively, and then we insert integron aadA2 of gene cassette, the recombinant plasmid was named pACINAD these two compatible restructuring copy number and the total integration of the sub-copy number plasmid was transformed into E. coli BL21 (DE3), After overnight incubation with fluorescent quantitative PCR technology the measured integral role in the integration of the sub-former divided by the latter is the integration of the frequency. Results in a host, E. coli BL21 (DE3) as measured by the integration of frequency of 1.87 × 10 -4 , the CV value of 24.6%, while in the background in the case of the absence of the high expression of the integrase enzyme integration, frequency lower than the 5.23 × 10 -8 . The method of establishing and laid the foundation for subsequent in-depth study integron capture the regulatory mechanism of gene cassette. Host factor for integration efficiency impact study of existing data suggest that the integration of the sub in the process of capturing gene cassettes may have other host factors participate in yet do not have the specific study. For this purpose we use a transposon, containing both of recombinant plasmid pUCINT pACINAD E. coli BL21 (DE3), create insertion mutant library. This mutation library screening using the aforementioned methods, to find an integrated frequency increased 26-fold compared to the corresponding wild-type strain, mutants, named the TIM. Differentially expressed gene screening use of coli gene chips the mutant and its corresponding wild-type strain the results detected differences in gene expression (52), including 33 up-regulated genes and 19 down-regulated genes. The involves the original phage DLP12 Total eight, the expression of these genes were up, they were ybcT, peaD 'ybcW exoD' ybcX, nohB, renD ', ybcV. This aroused our attention, because existing data also indicate that the prophage exists in many bacterial chromosomes, we study the integration of sub-equally important tools for bacterial genome evolution. Fluorescence quantitative PCR to validate the expression levels of some of these genes, consistent with the results of gene chip detection data. Further studies showed that the ybcV, ybcW, and nohB gene has a certain role in promoting the integration reaction. 3. Gene cassette length for the impact of the integration of efficient So far, the exact mechanism of integrase-mediated gene cassette excision and integration role is still not clear. Integron clinical use of the vast majority of antibiotic resistance genes, however, about the length of the gene cassette rarely exceed 1kb. Therefore, we assume that the length does not exceed 1 kb gene cassette optimum substrate for the recombination reaction is catalyzed by the integrase. To this end, we constructed a series of carrying the same attC bit point, but a different length gene cassette recombinant plasmid. In the first part of the paper created by the method basis, measured on the above-mentioned different lengths of the excision and integration of the gene cassette frequency catalyzed by the integrase enzyme. Results show that the the excision frequency fluctuations between 3.08 × 10 -2 and of 1.08 × -2 , while not the integrase high expression background frequency of less than 2.38 × 10 -7 . By sequence determination of the PCR product, removal occurs by the recombination reaction of two the attC bit point. These data show little difference integrase enzyme mediated excision frequencies for the different length of the gene cassette. The frequency of excision of the gene cassette only in plasmid pACZC carry carried on a plasmid pSA24 the excision of the gene cassette the frequency of 2.4 times, while the length of the latter gene cassette is about seven times the length of the former gene cassette. Reflects the integration of the excision reaction catalyzed by the enzyme is not sensitive to the change in length of the gene cassette. The frequency fluctuation of the integration of the gene cassette of the above-mentioned different lengths between 8.18 × 10 -5 and of 1.39 × -6 , with the increase of the length of the gene cassette, integrated with frequency significantly reduce the length of the gene cassette integrase enzyme catalyzed integration efficiency greater impact. These findings suggest that, integration of their rate-limiting step in the process of integron capture gene cassettes. Meanwhile, we prove that with the increase in the length of the gene cassette gene cassette excision frequency only slightly reduced integration frequency is a significant reduction. Reflects the recombination reaction catalyzed by the integrase enzyme molecule than the intermolecular recombination reaction is much easier, The a long gene cassette may affect the stability of the complexes formed between the integrase enzyme and its DNA substrate. Antibiotic concentration for the integration efficiency integron to capture gene cassette is random, under strong selection pressure in the environment, and ultimately get the most conducive to the survival of the host bacteria gene cassette arrangement and the composition of the bacteria are retained. Isolated in clinical integration sub in the vast majority of the gene cassette, are coding for known antibiotic resistance, which may be provided with antibiotics widely used in clinical. But so far also without integration enzyme catalyzed recombination reaction efficiency and the relationship of the concentration of antibiotic reported. In this regard, we have contained in the third position a aadA2 gene cassette integrated sub-cloned into the plasmid pACYC184 by. The recombinant plasmid was transformed into high expression integrase enzyme of the E. coli BL21 (DE3) after the addition of different concentrations of streptomycin After overnight culture, using the method established in the first part of the thesis Determination of aadA2 gene cassette reorganization in attI loci efficiency. The results show that in the case of the absence of the high expression of the integrase enzyme background frequency of less than 1.75 × 10 -7 . In the case of high expression of the integrase enzyme, aadA2 gene cassette can the integration sub attI locus recombination occurs, but the recombination frequency change, with the streptomycin concentration increased integration frequencies from 1.97 × 10 -3 < / sup> increased to 1.32. That a certain concentration of antibiotics can affect the integration frequency. Further confirmed by Western blotting, the expression levels of the of aadA2 gene the cartridge attI sites about 10 times in the third position of the expression level of integration of the sub-. Of enzyme expression level of integration for the integration efficiency as previously described in a certain range to improve integration of the expression levels of the enzyme can make the integration frequency is significantly improved, but, on this basis, to further increase the expression level of the integrase enzyme, whether cause integration frequency is further increased? we measured the frequency of integration of the gene cassette was cloned in the plasmid pUC19, integrase gene in different concentrations of IPTG induction, the catalyzed. The measured results are shown in a different inducing agent concentration, integrating the frequency and no significant changes. In order to further confirm this phenomenon, we also measured the frequency of integration of the gene cassette was cloned in the pET28a plasmid integrase gene in different concentrations of IPTG induction, the catalyzed. The measurement results show that different inducer concentrations, the integration of the frequency of the same no significant changes. SDS-PAGE showed cloned in pET28a plasmid integrase gene induced by IPTG, was significantly improved, and finally confirmed by Western blotting. This shows that the expression level of the integrase enzyme not decide they mediated integration reaction efficiency, may also be other factors such as the overall level of the gene cassette attC sites exist in the form of a single-stranded, integration the enzyme tetramer stability and so on, integration reaction also have a certain effect.

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