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학위논문 상세정보

Development of Biosensors by Engineering the Two Component System 원문보기

  • 저자

    Irisappan Ganesh

  • 학위수여기관

    울산대학교

  • 학위구분

    국내박사

  • 학과

    화학공학전공

  • 지도교수

    홍순호

  • 발행년도

    2014

  • 총페이지

  • 키워드

  • 언어

    eng

  • 원문 URL

    http://www.riss.kr/link?id=T13540253&outLink=K  

  • 초록

    In an attempt to develop a high throughput screening system for microorganisms producing high amounts of valuable molecules, chimeric two component system (TCS) was constructed. Chimeric two component system was considered to be an ideal candidate for employing the expression of the gfp gene under the respective gene promoter in aerobic condition, in which the gfp expression level is expected to be proportional to the concentration of metabolites present in the medium. However, in nature E. coli TCS could not produce certain signals which are strong enough to mediate the expression of GFP responding various molecule concentrations. Thus TCS from same or other bacterial species which can able to sense biochemical's was modified by recruiting the EnvZ/OmpR system, the most-studied TCS in E. coli that regulates the expression of outer membrane porins, OmpF and OmpC. As a result, four chimeric TCS was constructed (DcuSZ, MalKZ, AauZ and MoxYZ for sensing fumarate, malate, acidic amino acids and methanol respectively) by fusing the sensor histidine kinase of unique TCS (DcuS, MalK, AauS and MoxY) with the cytoplasmic catalytic domain of EnvZ, in which the expression of the gfp gene or the ompC gene was mediated by the ompC gene promoter through the cognate response regulator, OmpR. In case of chimeric DcuSZ, the output signals produced by the chimeric DcuSZ TCS were superior to those of the former DcuS/DcuR TCS in aerobic condition in the presence of fumarate. Real time quantitave PCR analysis of the ompC gene expression and the analysis of the fluorescence of GFP suggested that the level of the expressions of the ompC gene and the gfp gene increased as the fumarate concentration increased. Moreover, principal component analysis with other C4-dicarboxylates showed that DcuSZ chimera was highly specific to fumarate and other C4-dicarboxylates. Similiarily with MalkZ, the output signals produced by the chimeric MalKZ TCS were strong and superior. Real-time quantitative PCR and GFP fluorescence studies supports the above phenomenon by increasing the ompC gene expression and GFP fluorescence,when malate concentration increased. Moreover, principal component analysis with other C4-dicarboxylates showed that MalKZ chimera was highly specific to malate. To understand the feedback synergy and the signal transduction kinetics between MalKZ, DcuSZ and OmpR, a transcriptional feedback loop network was also characterized. The AauS/AauR two component system controls expression of a number of proteins including periplasmic glutaminase/asparaginase and ATP- binding cassette for the metabolism of acidic amino acids in Pseudomonas putida KT2440. AauS is a sensor kinase that ultimately controls aau gene expression through its cognate response regulator, AauR. The AauS/AauR two component systems were not found in Enterobacter such as Escherichia coli except in Pseudomonas putida KT2440. In this study, for sensing cellular glutamate, a new pathway was designed and constructed. Here EnvZ?E. coli strain was used as a chassis, and constructed two different devices: a sensor and an actuator assembled using OmpR as a standardized mediator. The AauZ was built by the fusion of the periplasmic and transmembrane domains of AauS protein with an EnvZ kinase domain. When the receptor complex binds AauS, an allosteric motion is propagated to the cytoplasmic EnvZ kinase domain, resulting in autophosphorylation and subsequent phosphate transfer to the OmpR transcription factor, which finally induces transcription of the ompC promoter. Transcriptional analysis of gfp fusions containing the ompC promoter confirmed that their expression of gfp is indeed induced by the chimeric system in the presence of acidic amino acids. In Paracoccus denitrificans, MoxY/X is a sensor kinase/response regulator that controls the expression of the moxF gene in response to formaldehyde which was formed from methanol. In an attempt to develop a high throughput screening system in Escherichia coli, to screen microorganisms producing high amounts of methanol, MoxY/MoxX TCS was modified by recruiting the EnvZ/OmpR system. A chimeric MoxY/EnvZ (MoxYZ) TCS was constructed by fusing the sensor histidine kinase of MoxY with the cytoplasmic catalytic domain of EnvZ, in which the expression of the gfp gene or the ompC gene was mediated by the ompC gene promoter through the cognate response regulator, OmpR. Real time quantitative PCR analysis of the ompC gene expression and the analysis of the GFP fluorescence suggested that the level of the expressions of the ompC gene and the gfp gene expression was increased up to 0.01% of methanol induction and decreased as the methanol concentration increased. These low levels of sensing may help to ensure a rapid response to small amounts of adventitiously formed methanol. The chimeric strategy described here should be a good platform to develop various chimeric TCS-based bacteria biosensors that can be used for the development of engineered microorganisms for the production of valuable biochemicals.


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