自動誘導(dǎo)超級肉湯培養(yǎng)基 Super Broth

自動誘導(dǎo)超級肉湯培養(yǎng)基Super Broth產(chǎn)品描述：

自動誘導(dǎo)超級肉湯培養(yǎng)基(Autoinductive Super Broth)也稱自動誘導(dǎo)SB，用于大腸桿菌Lac啟動子驅(qū)動的的重組蛋白表達，無需添加IPTG。自誘導(dǎo)培養(yǎng)基能夠獲得高細(xì)胞密度，蛋白表達率高出IPTG誘導(dǎo)過程好幾倍。

SB自動誘導(dǎo)培養(yǎng)基工作原理

SB培養(yǎng)基由于其高密度的營養(yǎng)成分，非常適合重組大腸桿菌的快速生長。超級肉湯培養(yǎng)基是一種加富培養(yǎng)基，配方基于TB培養(yǎng)基（Terrific Broth），后者由LB培養(yǎng)基改進而來，目的是達到高細(xì)胞密度。E. coli在該培養(yǎng)基中生長速度快于其在LB培養(yǎng)基或 2x YT肉湯中的生長速度。SB培養(yǎng)基被認(rèn)為是很佳的質(zhì)粒擴增培養(yǎng)基。一般來說，在液體培養(yǎng)基中大腸桿菌細(xì)胞生長的質(zhì)量如下：LB Broth < 2x YT Broth < Terrific Broth < Super Broth。

該培養(yǎng)基中含有理想比例的葡萄糖和α-乳糖作為碳源和能量來源。胰蛋白胨提供氮源、維生素、礦物質(zhì)和氨基酸，這些營養(yǎng)成分是細(xì)胞生長所必需的。酵母提取物是豐富的B族維生素來源。Studier salts為細(xì)菌細(xì)胞的生長提供很佳的生理環(huán)境。

通常，外源蛋白在細(xì)菌中表達時常使用IPTG誘導(dǎo)的啟動子，如Lac啟動子。當(dāng)細(xì)胞密度達到理想值時，通過向培養(yǎng)基中加入IPTG的方法誘導(dǎo)蛋白表達。使用這個自動誘導(dǎo)培養(yǎng)基，不再需要監(jiān)測細(xì)胞密度和添加IPTG。葡萄糖作為半乳糖操縱子的阻遏因子阻止細(xì)菌利用α-乳糖，而被優(yōu)先代謝，促進高密度生長。一旦培養(yǎng)基中的葡萄糖被耗盡（通常發(fā)生在對數(shù)期的后期），乳糖被?-半乳糖苷酶轉(zhuǎn)換成異乳糖（葡萄糖-1,6-半乳糖），而后者作為IPTG誘導(dǎo)型啟動子的誘導(dǎo)劑，引起乳糖阻遏子從與DNA結(jié)合的位點上釋放，啟動重組蛋白的表達。對于DE3基因型的E.coli中，異乳糖使T7lac啟動子去阻遏，并誘導(dǎo)lacUV5啟動子表達T7 RNA聚合酶。通過這種方式，在細(xì)菌培養(yǎng)物生長到某一特定點時蛋白表達自發(fā)開始，去掉了監(jiān)測細(xì)胞密度（OD600）和添加IPTG這兩個步驟。與傳統(tǒng)IPTG誘導(dǎo)的蛋白表達過程相比，使用自動誘導(dǎo)培養(yǎng)基極大地方便和簡化了試驗流程。

使用步驟：

1. 稱取74.85 脫水培養(yǎng)基粉末，用1L去離子水加熱溶解。

2. 煮沸1min。

3. 115°C 滅菌20min。

不要過度加熱，避免乳糖分解和培養(yǎng)基顏色加深。冷卻后保存在2-8oC，如果不染菌，可以保存2個月左右。

Quantity: 500g

Appearance: Beige powder. Autoclaved medium should be amber.

Storage: 2oC – 25oC. When not in use, keep container closed to avoid hydration.

配方Formulation (g/L)

Tryptone: 35.00

Yeast Extract: 20.00

MgSO4: 0,15

(NH4) 2SO4: 3,30

KH2PO4: 6,80

Na2HPO4: 7,10

Glucose 0,50

Alpha Lactose 2,00

Final pH (25oC): 7,0 ± 0,2

配置Preparation:

Add 45,85g of the dehydrated medium to one liter of distilled water. Mix well and dissolve by heating with regular agitation. Boil for 1 minute in order to dissolve completely. Dispense in appropriate containers and sterilize by autoclaving at 121oC for 15 to 20 minutes. Store at 2oC to 8oC.

使用Usage

Commonly, heterologous protein expression is carried out in bacterial systems where the expression is under the control of an IPTG-inducible promoter, such as the Lac promoter. Cells are grown until a desired density and protein expression is subsequently induced by adding IPTG to the medium. With this Auto Induction Medium (AIM), it is no longer required to monitor cell density and to add IPTG at the proper stage, as the medium contains an optimized ratio of glucose and alpha lactose as carbon sources. Glucose, who serves as a repressor of the Lac operon, by preventing uptake of alpha lactose (hence and IPTG) is metabolized preferentially during growth, promoting high cell density. Once glucose is depleted, usually in mid to late log phase, lactose enters the cell where it is converted by ?galactosidase into allolactose, which in turn serves as the inducer of the IPTG-inducible promoter, resulting in protein expression. This is a great convenience and simplifies manual or automatic induction and analysis of multiple clones compared to conventional IPTG  induction.