基因迴圈功能之演化穩定度研究

Abstract

合成生物學是現代生物學的新興研究領域，其利用基因重組技術讓基因迴路依循可預期的方式在細胞中表現出特定功能，控制生物體進行一系列的工作。現今已發表的基因迴路面臨的難題是基因迴路在細胞內表現短暫功能後會逐漸崩潰，無法穩定運作於細胞中，喪失實用價值。喪失基因迴路功能的突變菌株可以減少宿主的代謝負擔，使突變菌株有生長速度的優勢，在多代繁殖後，突變菌株將會佔細菌族群的多數。為了要測量代謝負荷和演化穩定度之間的相關性，本研究建構了名為T9002M的質體，此質體具備群體感應功能且可被acyl-homoserine lactone（AHL）所誘導。我們可藉由AHL的濃度控制T9002M質體的表現強度，使宿主細胞產生不同的代謝負擔藉由持續測量100代宿主中T9002M質體的螢光強度表現，發現螢光強度隨著細菌世代的演進出現了顯著的下降。為了定量基因迴路的演化穩定度，本研究提供了一個動態模型用以計算基因電路功能的穩健性和質體功能的半衰期。此外，在突變的T9002M質體中發現插入轉位子的突變、點突變和缺失突變。研究中應用了兩種策略減少突變與增進演化穩定性。策略一是將recA基因從大腸桿菌中剔除，這樣可以避免同源重組相關的突變發生，例如缺失突變。策略二則是在宿主中表現單股DNA結合蛋白，可以穩定複製時產生的單股DNA，避免複製滑動相關的突變發生。透過recA基因剔除的宿主與表現單股DNA結合蛋白，本研究成功地延長了基因電路的演化穩定度。實驗中也測量了T9002M在大腸桿菌菌株DH10B、DH5α、BL21和JM109中的演化穩定度。在不同的宿主中，JM109具有更長的半衰期和更好的穩健性。此外，在相同的初始螢光強度條件下，recA基因剔除的大腸桿菌也有更長的半衰期，而且其更具有表現多量蛋白質的優點。因此，JM109與recA基因剔除的大腸桿菌是適合基因電路表現的宿主。

The main goal of the nascent field of synthetic biology is the design and construction of biological systems with the desired behavior. The construction of biological synthetic circuits has become feasible through synthetic biology. However, evolutionary stability is a problem in the designed plasmids if there is no selective pressure to maintain desired functions. Loss-of-function mutants often have a growth advantage because a mutation that inactivates a designed plasmid can reduce its metabolic burden. To measurement the correlation between the metabolic load and evolutionary stability, an acyl-homoserine lactone (AHL)-induced quorum sensing plasmid, named as T9002M, was constructed. Using this plasmid, we can control the metabolic load by the concentration of AHL and measure the GFP fluorescence intensity which decreased with E.coli generations. The fluorescence intensity of T9002M plasmid propagated in E. coli 100 generations was measured, and we found the fluorescence intensity of a GFP expression plasmid decreased dramatically with bacteria generations. To characterize the evolutionary stability of genetic circuits, a dynamic model was provided to calculate the functional robustness and half-life of plasmid. The insertion of transposons, point mutation and deletion were found in designed circuits. To improvement the evolutionary stability, two strategies applied. A recA gene was knocked out from E.coli strain to avoid homologous recombination, and the single strain binding proteins (SSB) was expressed in host bacteria to avoid replication slippage. By utilize recA deletion host and co-expression of SSB protein, the stability of circuit successfully lengthened and had more evolutionary stability. Evolutionary stability of T9002M in E.coli strains DH5α, DH10B, BL21 and JM109 was also measured. JM109 have the longer half-life and better robustness among the different hosts. Additionally, the recA-deleted E.coli has an advantage for protein expression, and also has longer half-life in the same initial fluorescent intensity. JM109 and the recA-deleted E.coli are suitable hosts for genetic circuits.