轉錄對於大腸桿菌內多套數質體聚集行為的影響

The effects of transcription on the clustering behavior of the high-copy-number plasmids in Escherichia coli cells.

葉懷澤(Ye Huai-Ze)

指導教授 ： 張宜仁

國立臺灣師範大學/理學院/物理學系/碩士(2019年)

https://doi.org/10.6345/NTNU201900852

摘要

多套數的質體在大腸桿菌內並非隨機分佈於整個細胞，而在特定位置存在著 群集(聚類)。根據先前研究，其聚類原因可能來自轉錄造成。我們以單分子追蹤 與影像分析來觀察質體聚類行為，在單分子追蹤實驗，藉由螢光顏色區分，在質 體群集中限定一螢光標記質體為單一質體，並觀察單一質體的動態行為。我們使 用多套數質體 ColE1 的衍生質體 pBR322 做為實驗對象，並設計兩組具有相同複 製起點，但不同螢光抑制操作系統(Fluorescent repressor operator system)標記的質 體，並將兩種不同的質體植入到 BW25113。且使用抑制操作配對(PhlF-PphlF)作為 RNAII 的調控，以達到複製質體數量控管;另外，為了抑制轉錄行為，我們亦使 用了抑制操作配對(QacR-PqacR)作為抗藥性基因轉錄行為調控的手段。總而言之， 我們的研究表明多套數質體的確會受到轉錄行為抑制而導致聚類行為減弱甚至 消失。

關鍵字

多套數質體 ； 轉錄抑制

並列摘要

Rather than randomly distributed, the high-copy-number (hcn) plasmids exhibit as clusters at specific cellular regions in Escherichia coli cells, and the transcription of their encoded genes has been considered as one of the main reason behind this phenomenon. To verify this hypothesis, the responses of the clustering behavior of hcn plasmids induced by various transcription strengths were investigated via the global distribution of the plasmids in cells and the single molecule tracking on single plasmids. To visualize the plasmids and to identify a single plasmid, two ColE1-derivative plasmids which were tagged by two different fluorescence colors via different Fluorescent repressor-operator system (FROS) and encode different genes of antibiotic resistance proteins were co-transformed into the Escherichia coli strain BW25113. A repressor-operator pair, PhlF-PphlF, was used to control the copy number of one of the plasmids by regulating the expression of the primer RNAII of the plasmid replication. Additionally, another repressor-operator pair, QacR-PqacR, was applied to regulate the transcription of the genes encoding the antibiotic resistance proteins, which is the main expression of the plasmids. As a result, the clustering behavior of the hcn plasmids became weak or disappeared, when the transcription of their encoded genes was inhibited.

並列關鍵字

high-copy-number plasmids ； transcriptional repression

參考文獻

Kado, C.I., Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens. Frontiers in microbiology, 2014. 5: p. 340.

Tolmasky, M. and J.C. Alonso, Plasmids: biology and impact in biotechnology and discovery. 2015: ASM Press.

Shi, J. and D.P. Biek, A versatile low-copy-number cloning vector derived from plasmid F. Gene, 1995. 164(1): p. 55-58.

Nordström, K., Plasmid R1—replication and its control. Plasmid, 2006. 55(1): p. 1-26.

Figurski, D.H., R.J. Meyer, and D.R. Helinski, Suppression of ColE1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro. Journal of molecular biology, 1979. 133(3): p. 295-318.