Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51124
標題: I. 枯草桿菌Bacillus subtilis DB104電轉形效率之增進 II. 設計、合成第一型抗凍蛋白基因及其於枯草桿菌、大腸桿菌中之表現
I. Improvement of the electrotransformation efficiency of threonine-treated Bacillus subtilis DB104. II. Design and synthesis of type I antifreeze protein gene and expression in Bacillus subtilis and Escherichia coli
作者: 王志鵬
Wang, Jyh-perng
關鍵字: Bacillus subtilis;枯草桿菌;electrotransformation;antifreeze protein;Escherichia coli;電轉形;抗凍蛋白;大腸桿菌
出版社: 食品科學系
摘要: 
第一部分
枯草桿菌Bacillus subtilis DB104電轉形效率之增進
中文摘要
枯草桿菌Bacillus subtilis 為發酵工業上重要之菌種,亦為遺傳工程上常用之宿主細胞(host cell)。為有效進行菌種改良或將外源基因(foreign gene)導入枯草桿菌中進行表現,轉形(transformation)是常用之手段。目前見諸於報導的枯草桿菌轉形法,有自然轉形法(natural transformation)、原生質體轉形法(protoplast transformation)、電轉形法(electroporation;electrotransformation)及粒子槍(particle inflow gun)導入法。其中電轉形法之操作較為簡單、省時。但枯草桿菌利用此法進行轉形時,轉形效率普遍不高,約在103~104 transformants / μg DNA。本研究之主要目的在於增進Bacillus subtilis DB104電轉形流程之效率。以作為後續研究之基礎。研究指出枯草桿菌之細胞壁經弱化後,可使轉形效率提昇。弱化細胞壁之方法有利用溶菌處理菌體,或於生長培養基中添加甘胺酸(glycine)或酥胺酸(threonine)等。本研究主要採用1 % 之酥胺酸(threonine)添加於生長培養基中,先藉以弱化Bacillus subtilis DB104之細胞壁。而後探討不同的因子,包括:電的因子(電壓、電阻)、細胞濃度、質體濃度、質體純度、質體來源、電轉形溶液、再生培養基及再生時間對此細胞轉形效率之影響。結果顯示:(1)於電場強度8.75 kV/cm、電阻500Ω之條件下進行電轉形,可獲得較佳之電轉形效率。(2)轉形效率會隨菌體濃度之增加而增加。(3)質體之濃度對轉形效率之影響不大,然而質體之來源與純度卻有影響。(4)最佳的電轉形緩衝液為SHMPYT(0.25M sucrose,1mM Hepes,1mM MgCl2,20%(v/v) PEG (polyethylene glycol) 6000,0.125% yeast extract,0.25% tryptone)。(5)最佳之再生時間為2小時。(6)最佳之再生培養基為2LB。於較佳之條件下進行電轉形,轉形效率最高可達2.25×105 transformants / μg DNA,較原始流程之效率7.22×103 transformants / μg為佳。轉形株內之質體經抽出確認,發現質體之大小、型態並未改變。且此修飾流程可直接將黏合混合物(ligation mixture)轉形入Bacillus subtilis DB104中。
第二部分
設計、合成第一型抗凍蛋白基因及其在枯草桿菌、大腸桿菌中之表現
中文摘要
抗凍蛋白(antifreeze proteins)為某些生存於極地或寒冷地區之生物體內之蛋白質。其具有降低生物體體液凍結點之效果,能使生物避免凍結而死亡。此外,研究顯示抗凍蛋白還具有修飾冰晶構形使冰晶變小及抑制冰晶再結晶之特性。此兩特性使得抗凍蛋白在細胞組織冷凍保存及冷凍食品品質保持上具有很大的應用潛力。
目前以魚類來源的抗凍蛋白被研究的較為清楚,其主要有五種種類:抗凍醣蛋白(antifreeze glycoproteins)、第一型抗凍蛋白(type I antifreeze proteins)、第二型抗凍蛋白(type II antifreeze proteins)、第三型抗凍蛋白(type III antifreeze proteins)及第四型抗凍蛋白(type IV antifreeze proteins)。其中第一型抗凍蛋白之結構較為簡單、分子量較小且相關之結構-功能研究甚多。
本研究主要在於設計、合成第一型抗凍蛋白基因,並嘗試於枯草桿菌及大腸桿菌中表現抗凍蛋白,以期未來能加以運用。首先以比目魚第一型抗凍蛋白HPLC6之結構功能特性相關研究為基礎,於蛋白質層次上設計第一型抗凍蛋白。重組第一型抗凍蛋白設計含有四個重複的胺基酸單元(Thr-X2-Asx-X7),其C端並設計加入6個組胺酸(histidine),以利重組抗凍蛋白之純化。若以融合蛋白之形式表現抗凍蛋白,則於蛋白質N端加入天門冬胺酸(aspartic acid)-脯胺酸(proline)序列,未來可利用甲酸(formic acid)處理去除掉蛋白質N端之融合夥伴(fusion partner)。之後將蛋白質序列推衍為DNA序列,設計引子利用重疊延展聚合連鎖反應(overlap extension polymerase chain reaction)合成抗凍蛋白基因。將合成之抗凍蛋白基因嵌入枯草桿菌及大腸桿菌載體中,分別轉形入枯草桿菌及大腸桿菌宿主細胞中,進行表現。於枯草桿菌中,主要欲以Subtilisin YaB signal peptide-抗凍蛋白及Subtilisin YaB signal peptide-propeptide-部分Subtilisin YaB胺基酸序列-抗凍蛋白等兩種融合蛋白質形式表現重組抗凍蛋白。但這兩種形式之抗凍蛋白表現量皆不佳,可能與基因表現所利用之Subtilisin YaB啟動子有關。於大腸桿菌中,主要利用pQE表現系統及pET表現系統來表現抗凍蛋白。於pET表現系統中,並探討Subtilisin YaB及Omp T蛋白之signal peptide對重組抗凍蛋白分泌之影響。結果顯示:於IPTG誘導後,各種表現系統皆可表現重組抗凍蛋白。其中驚訝地發現帶有Subtilisin YaB signal peptide之抗凍蛋白能分泌至胞外。反而帶有同源性peptide~OmpT peptide之重組抗凍蛋白分泌能力不佳, 其主要於胞內形成包涵體(inclusion body)。表現之抗凍蛋白經Ni-NTA樹脂進行親和性純化及N端定序後,確認表現之蛋白質與設計之抗凍蛋白序列相吻合。且表現之抗凍蛋白具有減小冰晶之抗凍活性。

I.Part I. Improvement of the electrotransformation efficiency of threonine-treated Bacillus subtilis
Bacillus subtilis is an industrial important bacterium, which has been used for the manufacture of a variety of enzymes, antibiotics and fine biochemicals. For strain improvement and genetic manipulation, transformation of this bacterium is an important step. There are several methods to introduce plasmid DNA into Bacillus subtilis, such as competent cell transformation, protoplast transformation, and electrotransformation. Among these methods, electro-transformation is the most attractive approach for its simplicity and easiness. However, the transformation efficiency by electrotransformation is generally low. For this reason, many researchers are attempting to improve the transformation efficiency of electrotransformation.
In this study, we tried to improve the electrotransformation efficiency of Bacillus subtilis DB104. The cell wall of Bacillus subtilis DB104 is weakened by supplement of threonine in culture medium. We examined the effect of electrical parameters, cell concentration, the concentration of plasmid DNA, plasmid purity, plasmid source, electroporation buffer, recovery medium and recovery time on the electrotransformation efficiency of threonine-treated Bacillus subtilis. The results showed: (1)Efficiencies of transformation increased with applied voltage to an optimum of 1.77×104 transformants /μg at a field strength of 8.75 kV/cm and resistance of 500Ω. (2)The transformation efficiency increased with the increases of cell concentrations. (3)Plasmid concentration did not influence transformation efficiency, but the plasmid purity and source did. (4)The best electrotransformation buffer is SHMPYT(0.25M sucrose, 1mM Hepes, 1mM MgCl2, 20%(v/v) PEG (polyethylene glycol) 6000, 0.125% yeast extract, 0.25% tryptone). (5)The optimal recovery time is 2 hours. (6) The optimal medium is 2LB. The maximum transformation efficiency of Bacillus subtilis DB104 was 2.25×105 transformants/μg. It is much higher than the transformation efficiency of original procedure (7.22×103 transformants/μg). Transferred plasmid DNAs isolated from transformants were the same as those of intact plasmids. Therefore, it is clear that the transferred DNAs did not undergo significant rearrangement or deletion. Using this procedure, ligation mixture can be directly transformed into Bacillus subtilis DB104, allowing direct molecular cloning of DNA into this organism.
II.Design and synthesis of type I antifreeze protein gene and expression in Bacillus subtilis and Escherichia coli.
Abstract
Antifreeze proteins (AFPs) are a group of proteins that can depress freezing point, inhibit ice recrystallization and modify the morphology of ice crystal. They are found in a wide range of organisms living in cold ambient conditions, including bacteria, fungi, plants, invertebrates and fish. Among these proteins, fish antifreeze proteins have been studied extensively. To date, five distinct types of fish AFPs have been described: antifreeze glycoproteins (AFGP), type I antifreeze proteins (AFP I), type II antifreeze proteins (AFP II), type III antifreeze proteins (AFP III) and type IV antifreeze proteins (AFP IV). In order to study their structure-function relationship or to apply them in industry, it is essential to gain a great quantity of these proteins. Three methods were developed to achieve this purpose: chemical synthesis, genetic engineering and isolation from fish bloods. Among these methods, genetic engineering is the most attractive one for scientists.
In this study, we designed and constructed a synthetic gene for recombinant antifreeze protein( rAFP ). The protein sequence of rAFP was designed to include four copies of the 11 amino acid antifreeze motif (Thr-X2-polar amino acid-X7) and was reverted into nucleotide sequence by Bacillus subtilis preferred codon usage. Using overlap extension polymerase chain Reaction technique to synthesize rAFP gene. Then, the PCR product was cloned into the Bacillus subtilis expression vector or Escherichia coli expression vector, and was transformed into the host strains by electroporation. In B. subtilis DB430, the expression level of antifreeze protein is very low. In E. coli host cells, the rAFP can be expressed after isopropyl-β-D-thiogalactopyranoside (IPTG) induction. rAFP fused to Subtilisin YaB signal peptide can be exported to periplasmic space, even secreted to medium by signal peptide cleavage through secretion machinery. Otherwise rAFP fused to OmpT signal peptide accumulated as insoluble inclusion bodies in the E. coli host. Expressed rAFPs can be purified by using Ni-NTA affinity chromatography. The N-terminal amino acid seqence of purified protein confirmed the identity of the expressed and purified protein as rAFP. All puried rAFPs can decrease the size of ice crystal.
URI: http://hdl.handle.net/11455/51124
Appears in Collections:食品暨應用生物科技學系

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.