建構擴散受限制的相互抑制基因網絡

Abstract

Mutual inhibition 長久以來都被認為是建立細胞不對稱性的核心機制之一，而在細胞內建立濃度梯度是最關鍵的過程。過去有許多研究透過合成生物學的方法建構 toggle switch 系統，並觀察到記憶性的行為，但未曾觀察到有濃度梯度的形成，我們推測是因為蛋白質擴散的速率太快所導致，因此必須限制蛋白質的擴散速率才有可能觀察到濃度梯度的形成。因此我們挑選了正交性的抑制子來建立傳統的 toggle switch 系統，以及利用 DivIVA 建立擴散受限制的 toggle switch 系統，若上述基因建構完成，大腸桿菌表現的性狀與我們預期的相同，則會將質體送入非細胞的微滴系統中更進一步得觀察性狀的表現。在此論文中，我們測試了 TetR, SrpR, LmrA 和 lambda 等四種抑制子，並以他們構建了完整的 toggle switch 系統，但並非所有的基因組都表現出理想中的性狀，SrpR 啟動子的強度偏弱，不足以產生足量的下游基因產物，導致細胞無法呈現出 DivIVA 應有的雙極性狀，且 SrpR 抑制子會對大腸桿菌細胞造成極大的代謝負擔，甚至導致質體的基因突變，另外， lambda 抑制子也如同 SrpR 抑制子會產生大腸桿菌的代謝負擔，導致基因組的表現也出現異常，而以目前的結果看來 TetR-LmrA 這組基因組的性狀最接近我們理想中的型態，能表現出足量的下游基因產物，行成 DivIVA 應有的雙極性狀，TetR 抑制子和 LmrA 抑制子也都能有效得抑制其對應的啟動子，再者，其中的 TetR 基因組能被 IPTG 誘導起來並有記憶性的行為。我們推測系統目前可能存在的問題是抑制子表現量過高造成的代謝異常，未來研究方向會致力於降低質體的拷貝數與調整核糖體結合位點的強度去降低整體細胞中的代謝負擔，使細胞表現出我們預期中的性狀。

Mutual inhibition has long been considered to be one of the central mechanisms for the establishment of asymmetry in cells, and the establishment of concentration gradients in cells is the most critical process. In the past, many studies have used synthetic biology methods to construct toggle switch and observed memory behavior, but the formation of concentration gradients has not been observed. We speculate that the rate of diffusion is too fast and it must be limited. We therefore picked orthogonal repressors to build traditional toggles and DivIVA as the fusion protein to build diffusion-limited toggle systems. If all of these genetic constructs in E.coli form the expected characteristics, we will put these plasmids into the microdroplet system to further observe the spatial patterns. In this thesis, four repressors, TetR, SrpR, LmrA and lambda, were tested and used to construct a complete toggle system. However, not all genetic circuits exhibited the expected behaviors. The strength of the SrpR promoter was too weak to produce sufficient downstream gene products, so that cells could not form the bipolar characteristics that DivIVA should have. In addition, the SrpR repressor caused a great metabolic burden in E. coli, which even led to plasmid mutation. On the other hand, the lambda repressor also caused metabolic burden in E. coli like the SrpR repressor, resulting in abnormal performance. Based on the current results, it seems that the traits of the TetR-LmrA pair were closest to our ideal type, which could produce sufficient downstream gene products to form the bipolar characterization of DivIVA. TetR repressor and LmrA repressor could also effectively inhibit their corresponding promoters. Furthermore, the TetR genetic circuit could be induced by IPTG and have memory behaviors. We speculate that the current problem of our system is the metabolic burden caused by the overexpression of the repressors. Our future research direction will focus on reducing the copy number of the plasmid and adjusting the strength of the ribosome binding site to reduce the metabolic burden in the cell.