利用濃度梯度產生器晶片培養並觀察藍綠菌PCC 7942品系於BG-11培養液和營養源的生長情形並找出最佳化條件

Using Concentration Gradient Generator Chip to Cultivate, Observe Growing Phenomenon of Cyanobacteria PCC7942 in BG-11 Medium and Nutrients

楊智竣(Yang, Chih Chun)

指導教授 ： 饒達仁

國立清華大學/工學院/奈米工程與微系統研究所/碩士(2015年)

摘要

藍綠菌，又稱為藍綠藻。在地球上已存在約35億年，是目前以來發現到最早的光合產氧生物。而此研究用的藍綠菌PCC 7942則是為其中一品系。藍綠菌的萃取物除了是現今生技產業很著名的保健食品外，也是化工和生物科技界所謂的績優股。因為容易進行基因轉殖並大量培養，可以產生很多替代能源。培養藍綠藻的水容液BG-11由10種成分組成，其配方自從公開發表後，濃度就一直是固定的。但很多研究員都試著改變各成分的濃度，想找到更適合藍綠菌成長的環境，縮短成長周期，或是增加細胞分裂速度。然而10種成分，若是每一次都只改變一種成分，該種成分又配成好多種濃度，再分別放置燒瓶培養，不僅相當耗時，也很浪費實驗空間和藥品的量。幸運的是，微機電(MEMS)蓬勃發展，很多實驗都能縮小到在一片晶片上進行，如此可以縮小實驗空間和耗材成本。此實驗便利用微機電技術中的黃光微影製程，去做母模，並用軟微影技術用2層PDMS做出含有第一層的主流道和第二層的儲存槽，再利用氧電漿接合2層PDMS。此論文將運用1維的裝置，並利用樹狀分支的概念，從2個流道，分成3個流道，再從3個分支成4個，最後分支成6個，並延伸到儲存槽。如此一來，將兩種不同濃度的溶液從入口端打入，就能產生濃度梯度。此裝置稱為濃度梯度產生器。每一格儲存槽量約為840μl。有了此裝置一次就能產生6種不濃濃度的環境去培養藍綠菌，大幅壓縮實驗空間和成本。培養在儲存槽的藍綠菌，經過3天的生長，有些能用顏色差異看出生長優劣，但都要去量測吸光值(O.D.)，做為生長情形好或壞的判斷依據。此外，本實驗還探討除了改變固定成分的個別濃度對藍綠菌生長的情形外，我們還額外加入營養源，例如維生素和尿素去觀察是否有利於藍綠菌的生長表現。最後實驗部分會利用傳統藍綠菌搖瓶培養去驗證生長結果是否和我們在微流體晶片所培養下的藍綠菌有相似的生長趨勢。

關鍵字

藍綠菌 ； BG-11培養液 ； 濃度梯度產生器 ； PDMS ； 吸光值(O.D.)

並列摘要

Cyanobacteria are phylum of bacteria that obtain their energy through photosynthesis. They are also known as Cyanophyta. It is known as the most famous transformation model in microorganisms. BG-11 medium which is composed of nine ingredients and carbon sources with specifically combinatorial ratio is used to cultivate cyanobacteria. One approach to improve the growth speed of cyanobacteria in a laboratory setting is to optimize the cultivation medium and tailor the supplements according to the microorganism’s need. A general cultivation medium for S. elongatus PCC7942 named BG-11 had been developed and is a standard prescription. However, many biochemical researchers still want to figure out a new recipe to make a new concentration ratio of ten ingredients to let the cyanobacteria grow faster and efficiently. Nowadays, with the advanced of nano and micro technology techniques, many experiments can be conducted on the small area or chips with a little restriction especially in biochemical or biomedical experiments used the MEMS techniques. In experiments, we would like to know the influence of each ingredient to the growth effect of cyanobacteria, therefore, we used the UV lithography to fabricate the concentration gradient generator (CGG) mold and cultivated gene transfer cyanobacteria pcc7942 in PDMS made chambers by using a high throughput micro channel design. Compared to conventional methods by cultivated manually in large amount of medium in big beakers, we used the microfluidics concentration gradient system to cultivated automatically and only with small amount of medium in biochips. It saves the experimental costs and areas as well. In this chip, we can observe six results detected by absorbance value (Aλ) with different concentration in same ingredient at the same time in 1D chips. Besides, we also add the nutrients such as vitamin and urea to cultivate cyanobacteria to find out whether those exotic nutrients will stimulate the growth of cyanobacteria. Finally, we will show the growth data cultivated by traditional flask methods to certify the similar trend with the cultivation in microfluidics chips.

並列關鍵字

concentration gradient generator ； Cyanobacteria ； BG-11 medium

參考文獻

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