本研究針對插入外源基因korAB並表現異源酶2-酮戊二酸：鐵氧還蛋白氧化還原酶(2-oxoglutarate:ferredoxin oxidoreductase,OGOR)的大腸桿菌進行探討，該菌株展現出混營生長特性。在蘋果酸(malate)作為碳源以及硝酸鹽作為能量來源的條件下，這些大腸桿菌能夠吸收二氧化碳作為無機碳源。本研究基於前人研究工作，使用已經設計好的生物反應器，並結合經過第一階段適應性演化（以絲氨酸作為碳源）的菌株，展開第二階段適應性演化實驗。實驗以蘋果酸作為碳源，進行大約140天左右的演化過程。之後在整個演化過程中，利用生物反應器中的多項功能，例如：使用LED用於發射和接受偵測光的強度、無線傳輸模組等來實時監測和傳輸大腸桿菌在培養過程中的生長狀況，通過測量光學密度(600nm wavelength optical density,OD600)來記錄大腸桿菌的生長變化以及計算生長速率(growth rate)。這些數據不僅能揭示大腸桿菌在適應性演化過程中產生的生理變化還能幫助觀察演化產生的顯著性狀。在第二階段的適應性演化結束後，將以下四個不同階段的大腸桿菌進行對比實驗：1.剛植入外源基因korAB的大腸桿菌；2.尚未經歷以蘋果酸作為碳源的第二階段適應性演化但帶有korAB外源基因的大腸桿菌；3.已完成以蘋果酸作為碳源的第二階段適應性演化的大腸桿菌。4.未植入外源基因korAB的大腸桿菌。這些菌株分別在含有或者不含有硝酸鹽的培養基進行三重復實驗，並比較其在這些不同條件下的生長差異，從而探討適應性演化對其碳固定能力和代謝特性的影響

This study focuses on Escherichia coli engineered with the insertion of the exogenous korAB gene, which expresses the heterologous enzyme 2-oxoglutarate: ferredoxin oxidoreductase (OGOR). These engineered E. coli strains exhibit mixotrophic growth characteristics. Under conditions where malate is used as the carbon source and nitrate as the energy source, the bacteria can absorb carbon dioxide as an inorganic carbon source. Building on previous research, a custom-designed bioreactor was employed, and E. coli strains that had undergone the first stage of adaptive evolution (using serine as the carbon source) were subjected to a second-stage adaptive evolution experiment. In this stage, malate was used as the carbon source over a period of approximately 140 days.Throughout the evolution process, the bioreactor's variou and wireless transmission modules, were used to monitor and transmit real-time data on bacterial growth. The bacterial growth was tracked by measuring the optical density at 600 nm (OD600), and growth rates were calculated. This data not only revealed physiological changes in E. coli during the adaptive evolution but also helped observe significant traits arising from the evolution process.After the second stage of adaptive evolution, four different stages of E. coli were subjected to comparative experiments: (1) E. coli newly engineered with the korAB gene; (2) E. coli with the korAB gene but not yet subjected to the second-stage adaptive evolution using malate as the carbon source; (3) E. coli that completed the second-stage adaptive evolution using malate as the carbon source; and (4) E. coli without the korAB gene. These strains were cultivated in media with and without nitrate in triplicate experiments, and the growth differences under these conditions were compared. The goal was to investigate the effects of adaptive evolution on carbon fixation capacity and metabolic traits.s features, such as LED systems for emitting and detecting light intensity.

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