隨著生物資訊的蓬勃發展及各式基因編輯工具的建立，促使合成生物學的應用範

隨著生物資訊的蓬勃發展及各式基因編輯工具的建立，促使合成生物學的應用範圍更為廣泛，從傳統式表達單一蛋白的系統，拓展至代謝途徑的調控，使微生物成為生產各式化學品的細胞工廠。透過代謝途徑的調控來生產高濃度的化學品，仰賴微調控細胞的基因表現，若透過傳統的圖譜分析化學品生產則耗時又耗力，因此開發生物傳感器本位的偵測化學品生產，有利於優化化學品的產量。然而，生物感測器平台之困難點在於，對於某些化學品的感測，模式微生物無法具耐酸鹼性、耐熱性、高鹽或疏水性的環境，導致生物感測器的應用範圍受限。因此本計畫之目的在於提升微生物對不同環境及化學因子的耐受性，利用此菌株於不同的化學品感測，接著結合T7核糖核酸聚合酶/T7啟動子之調控因子，以建立小分子之高校生物感測器平台。最後，本計畫將建立一全新的大分子蛋白文庫篩選平台，由於目前高效的蛋白突變及篩選平台PACE仰賴操作兩種不同的微生物，導致技術門檻高而鮮少應用，我們藉由CRISPRi系統抑制大腸桿菌必須基因的表現作為篩選壓力來篩選不同蛋白在大腸桿菌的異源表達情況，此外引入蛋白突變文庫，更能篩選出高活性且利於模式生物異源表達的蛋白。

結果 (英文) 1:

With the establishment of the vigorous development of gene editing tools and various types of biological information, prompting the scope of application of synthetic biology is more extensive, from traditional single protein expression system, to expand the regulation of metabolic pathways of microorganisms to be produced all kinds of chemical cell factory products. Through the regulation of metabolic pathways to produce high concentrations of chemicals, cells rely on micro-regulation of gene expression, if the analysis of chemical production through the traditional pattern of the time-consuming and labor-intensive, so the development of biosensors to detect chemical production standard, it is advantageous to optimize the production of chemicals. However, the difficulty in that point biosensor platform for sensing certain chemicals, microorganisms can not model with acid and alkali resistance, heat resistance, high salt, or a hydrophobic environment, resulting in receiving application biosensor limit. The object of the project is to enhance the resistance of microorganisms to different environmental and chemical factors, the use of this strain was different chemical sensing, followed by T7 RNA polymerase binding / T7 promoter, the regulatory factor, in order to establish a small molecule the university biosensor platform. Finally, this project will build a new large protein library screening platform, due to the current operation of two different microorganisms and efficient protein mutation and screening platform PACE rely on, leading to high technical threshold and rarely applied, our system depression by CRISPRi E. coli expression gene must be screened different proteins in a heterologous expression in E. coli, a library of mutant proteins is introduced in addition, a high activity and better screened facilitate heterologous protein expression as a screening model organisms pressure.

結果 (英文) 2:

With the vigorous development of biological information and the establishment of various gene editing tools, the application of synthetic biology is more extensive, from the traditional system of expression of single protein, to the regulation of metabolic pathways, so that microorganisms become a cell factory for the production of various chemicals. The production of high concentrations of chemicals through the regulation of metabolic pathways, relying on the genetic performance of micro-regulated cells, and the time-consuming and labor-intensive analysis of chemical production through traditional maps, is conducive to optimizing the production of biosensor-based detection chemicals. However, the difficulty of the biosensor platform is that for the sensing of certain chemicals, the model microorganisms are not able to have an acid-resistant, thermal, high-salt or hydrophobic environment, resulting in limited application of biosensors. Therefore, the purpose of this program is to improve the tolerance of microorganisms to different environments and chemical factors, using this strain in different chemical sensing, and then combined with T7 RNA polymerase/T7 promoter regulatory factors to establish a small molecule of the university biosensor platform. Finally, the plan will establish a new macromolecule protein library screening platform, due to the current high-efficiency protein mutation and screening platform PACE rely on the operation of two different microorganisms, resulting in high technology and rare application, we use the CRISPCRISPRi system to inhibit the performance of E. coli must be gene as screening pressure to screen different proteins in E. coli heterogeneous expression, in addition to the introduction of protein mutation library, more able to screen out high activity and model of biological isogenous expression.

結果 (英文) 3:

With the rapid development of biological information and the establishment of various gene editing tools, the application of synthetic biology has been expanded from the traditional expression of single protein system to the regulation of metabolic pathway, which makes microorganisms become the cell factories for the production of various chemicals. The production of high concentration chemicals through the regulation of metabolic pathway depends on the fine-tuning of gene expression of cells. It is time-consuming and labor-consuming to analyze the chemical production through the traditional pattern. Therefore, the development of biosensor based detection chemical production is conducive to optimizing the chemical production. However, the difficulty of biosensor platform is that for the sensing of some chemicals, the model microorganism can not have the environment of acid-base resistance, heat resistance, high salt or hydrophobicity, which results in the limited application of biosensor. Therefore, the purpose of this project is to improve the tolerance of microorganisms to different environments and chemical factors, to use this strain to detect different chemicals, and then to combine the regulatory factors of T7 ribonucleic acid polymerase / T7 promoter, so as to establish a small molecular college biosensor platform. Finally, this project will establish a new screening platform for large molecular protein library. Due to the high efficiency of protein mutation and the fact that the platform pace relies on two different microorganisms, the technology threshold is high and the application is rare. We use crispri system to inhibit the expression of essential genes of E.coli as screening pressure to screen the heterologous expression of different proteins in E.coli, In addition, the introduction of protein mutation library can screen out proteins with high activity and favorable for heterologous expression of model organisms.