利用氣舉式發酵槽培養基因重組大腸桿菌生產colicin 1B之研究

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题名:	利用氣舉式發酵槽培養基因重組大腸桿菌生產colicin 1B之研究
其它题名:	The Study of Recombinant Protein-Colicin 1B Production in Escherichia coli by Using Airlift Bioreactor
作者:	狄雷薩 RESZA DIWANSYAH PUTRA
贡献者:	顏宏偉 YEN, HONG-WEI 化學工程與材料工程學系
关键词:	Escherichia coli;IPTG;SOS response;Protein Activity;Optical Density (OD) value
日期:	2019
上传时间:	2019-12-16T02:18:12Z (UTC)
摘要:	Escherichia coli (E. coli) is the most common host for recombinant protein production. A high cell density culture (HCDC) of E. coli is regarding be an efficient way to improve recombinant protein production, due to the low volume required, high efficiency, and relatively low cost of HCDC. To achieve HCDC, oxygen availability which is represented by dissolved oxygen (DO) is considered to be a crucial factor affects the production of recombinant protein in E. coli. In the other side, Induction step is crucial for the recombinant protein production. In the E. coli strain, there is a mechanism called SOS response.This mechanism takes the role to repair the DNA when it is damaged or the replication of DNA is inhibited. As a result of the DNA repair, the certain protein is produced in the biomass. This experiment conducted to produce colicin 1B protein which is usually used as antibiotics. E. coli biomass was cultured in 4 liters Luria Bertani (LB) medium inside the airlift fermenter. After the desired Optical Density (OD) value of the biomass was achieved, the mitomycin C was added as an inducer to give damage to the biomass. So, the SOS response mechanism was able to do recombinant protein production. Some variation conducted in this experiment were a variation of aeration, a variation of temperature, and variation of pH. In the variation of aeration, it showed that keeping low aeration after induction can increase the protein expression. This optimal aeration is 2 vvm before induction and 0.5 vvm after induction which obtain 214 protein expression. Besides that, it showed that the temperature did not give many changes to the protein expression which is stable between 211 and 212. Meanwhile, the acid or base condition is not good for the recombinant protein since the resulted protein expression is stable between 25 and 26 which is not as good as the neutral condition.Besides using the SOS response induction, the Isopropylthio-b-thiogalactopyranoside (IPTG) induction strain was also employed to evaluate the effect of the induction time point. The gene called T7, presenting in the pET vectors, is used for recombinant protein. When IPTG is present, the gene will activate the transcription of the desired protein. The evaluated induction time points are 0-hour, 5-hours and 24-hours. The result showed that the induction time point did not give effect to the protein activity. However, it gave effect to the result in cell concentration. The later induction time resulted in higher cell concentration since the metabolism resource was utilized in the growth phase.
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