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Aureobasidin A (金担子素),是从丝状 真菌 Aureobasidium pullulans No. R106 中分离出来的环酯肽类抗生素,是一种很强的抗真菌抗生素。在较低的浓度下 (0.1-0.5 μg/mL) 可对酵母产生毒性,对其敏感的真菌包括:酿酒酵母 (Saccharomyces cerevisiae)、裂殖酵母 (Schizosaccharomyces pombe)、光滑念珠菌 (Candida glabrata)、构巢曲霉 (Aspergillus nidulans) 和黑曲霉 (A. niger)。作用机制:AbA 抑制酵母中 AUR1 基因 (1,2) 编码的肌醇磷脂酰胺 (IPC) 合成酶的活性,AUR1 基因的突变形式 AUR1 C 的表达使酵母对 AbA 产生抗性。利用 AUR1-C 基因作为蛋白质互作的报告基因可降低仅用营养报告基因 (如 HIS3) 进行低严紧度初筛时产生的假阳性背景。由于 AbA 可以直接杀死敏感的酵母菌株,而不是只抑制菌株生长,所以基于 AbA 的筛选可降低酵母互作的假阳性。一般实际操作中,用低浓度的 AbA 进行初筛时会获得很多克隆,之后再提高筛选严谨度,比如用 4 个报告基因 (AUR1-C,HIS3, ADE2 和MEL1) 或提高 ABA 筛选浓度进行高严谨度的二次筛选以确认阳性克隆。
产品组分与配方:
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操作方法
1、Y2HGold (pGBKT7+pGADT7) 双杂系统:Y2HGold 菌株基因组中整合了 AbA 抗性基因 AUR1-C,Y2HGold 有四 个报告基因:AbAr,HIS3,ADE2,MEL1,当蛋白互作时会激活这四个报告基因的表达。酵母双杂的自激活验证和筛选试验中 AbA 的常规使用浓度为 125-200ng/ml。在做酵母双杂试验之前应对Bait质粒进行自激活验证,初始 AbA 浓度可以用 125ng/ml,若没有自激活,可将此浓度作为后面双杂试验时的筛选浓度,若有自激活可以提高 AbA 浓度,直到 Bait 质粒没有自激活为止,但 AbA 浓度最高不要超过 1000ng/ml (若超过此浓度仍然有自激活,说明酵母细胞中有内源转录因子或其他分子可以识别Bait质粒,激活 AUR1-C 基因,这种试验结果是不准确的)。 若酵母筛库试验中获得的阳性菌落太少,可以适当降低筛库时的AbA浓度到 100ng/ml。
2 、Y1HGold(pAbAi+pGADT7)单杂交系统:Y1HGold 菌株基因组本身没有整合 AbA 抗性基因 AUR1-C,在 pBait AbAi 经线性化整合到 Y1HGold 基因组后变成 Y1HGold[Bait/AbAi] 菌株,此菌株含有报告基因:AbAr,当蛋白与 DNA互作时会激活 AUR1-C (AbAr) 基因的表达。酵母单杂的自激活验证和筛选试验中AbA的常规使用浓度为 100- 200ng/ml。在做单杂试验之前应对 Bait 质粒进行自激活验证,初始 AbA 浓度可以设置为:100ng/ml\、150ng/ml、200ng/ml,若均没有自激活,可选最低浓度作为后面单杂试验时的筛选浓度,若有自激活可以提高 AbA 浓度,直到 Bait 质粒没有自激活为止,但 AbA 浓度最高不要超过 1000ng/ml (若超过此浓度仍然有自激活,说明酵母细胞中有内 源转录因子或其他分子可以识别报告基因,激活 AUR1-C 基因,这种试验结果是不准确的)。单杂的自激活验证可先将 菌液浓度调整到 OD600=0.2, 再稀释100倍到 0.002,取 100ul涂相应AbA浓度平板即可
3、 倒平板:酵母用固体培养基微波炉加热融化后或高压灭菌后冷却至55℃以下, 若配制 125ng/ml 的 AbA 平板,每 100ml 培养基加入 12.5ul 1mg/ml 的 Aureobasidin A 溶液混匀倒平板即可; 若配制 200ng/ml 的 AbA 平板,每 100ml 培养基加入 20ul 1mg/ml 的 Aureobasidin A 溶液混匀倒平板即可。
产品参数:
CAS:127785-64-2
英文名称:Aureobasidin A
分子量:1101.13
分子式:C₆₀H₉₂N₈O₁₁
纯度:≥98.0%
固体外观:白色粉末
溶解性:易溶于乙醇
注意事项
1、不开封的 Aureobasidin A 溶液(过滤除菌)可在-20℃保存两年,开封后若发现染菌,停止使用。
2、本产品仅供专业人员科研使用,不得用于临床诊断或治疗。
3、为了您的安全和健康,请穿实验服并戴一次性手套操作。
Aureobasidin A (AbA, also named Aureobasidin) is a cyclic depsipeptide antibiotic isolated from the filamentous fungus Aureobasidium pullulans strain No.R106, and it is a highly potent antifungal antibiotic. It is toxic to yeast at low concentrations ranging from 0.1 to 0.5 μg/mL. Fungi susceptible to this antibiotic include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida glabrata, Aspergillus nidulans and Aspergillus niger.Mechanism of Action AbA inhibits the activity of inositol phosphorylceramide (IPC) synthase encoded by the AUR1 gene in yeast. Expression of the mutant form of the AUR1 gene, AUR1-C, renders yeast resistant to AbA. Using the AUR1-C gene as a reporter gene for protein interaction assays can reduce the false-positive background that arises during low-stringency primary screening using only nutritional reporter genes such as HIS3. Since AbA directly kills susceptible yeast strains rather than merely inhibiting their growth, screening based on AbA helps decrease false positives in yeast interaction experiments. In routine experimental operations, a large number of clones can be obtained through primary screening with a low concentration of AbA. Afterwards, the stringency of screening is increased to confirm positive clones via high-stringency secondary screening, which can be done either by utilizing four reporter genes (AUR1-C, HIS3, ADE2 and MEL1) or by raising the screening concentration of AbA.
Product Composition and Formula:
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Operating Procedures
1. Y2HGold (pGBKT7 + pGADT7) Yeast Two-Hybrid System
The genome of Y2HGold strain is integrated with the AbA resistance gene AUR1-C, along with four reporter genes: AbAᵣ, HIS3, ADE2 and MEL1. Expression of these four reporter genes will be activated upon protein-protein interaction.
The routine working concentration of AbA for autoactivation verification and screening in yeast two-hybrid assays is 125–200 ng/mL.
Autoactivation verification of the bait plasmid must be performed prior to yeast two-hybrid experiments. An initial AbA concentration of 125 ng/mL is recommended. If no autoactivation is observed, this concentration can be adopted as the screening concentration for subsequent two-hybrid screening. If autoactivation occurs, increase the AbA concentration gradually until the bait plasmid shows no autoactivation. The maximum AbA concentration shall not exceed 1000 ng/mL. If autoactivation still persists above this concentration, it indicates that endogenous transcription factors or other intracellular molecules in yeast can recognize the bait plasmid and activate the AUR1-C gene, leading to unreliable experimental results.
If too few positive colonies are obtained during library screening, the AbA concentration for library screening may be appropriately reduced to 100 ng/mL.
2. Y1HGold (pAbAi + pGADT7) Yeast One-Hybrid System
The Y1HGold strain genome does not contain the integrated AbA resistance gene AUR1-C. After linearized pBait-AbAi plasmid is integrated into the Y1HGold genome, the engineered strain Y1HGold[Bait/AbAi] carrying the reporter gene AbAᵣ is generated. Expression of the AUR1-C (AbAᵣ) gene will be triggered when protein-DNA interaction takes place.
The standard AbA concentration range for autoactivation test and screening in yeast one-hybrid assays is 100–200 ng/mL.
Conduct autoactivation verification of the bait plasmid before one-hybrid experiments; set initial AbA concentrations at 100 ng/mL, 150 ng/mL and 200 ng/mL. If no autoactivation is detected at all three concentrations, the lowest valid concentration can be used for subsequent one-hybrid screening. Raise the AbA concentration stepwise to eliminate bait plasmid autoactivation if needed, with an upper limit of 1000 ng/mL. Persistent autoactivation beyond this limit implies endogenous yeast factors activate the reporter gene non-specifically, rendering experimental data invalid.
For one-hybrid autoactivation assay, adjust the bacterial suspension to OD₆₀₀ = 0.2, dilute it 100-fold to OD₆₀₀ ≈ 0.002, and spread 100 μL of the diluted suspension onto plates supplemented with corresponding concentrations of AbA.
3. Plate Pouring Instructions
Melt yeast solid medium by microwave heating or after autoclaving, then cool it down to below 55 °C before adding AbA.
To prepare plates with 125 ng/mL AbA: add 12.5 μL of 1 mg/mL Aureobasidin A stock solution into every 100 mL medium, mix well and pour plates.
To prepare plates with 200 ng/mL AbA: add 20 μL of 1 mg/mL Aureobasidin A stock solution into every 100 mL medium, mix well and pour plates.
Product Specifications:
CAS Number: 127785-64-2
English Name: Aureobasidin A
Molecular Weight: 1101.13
Molecular Formula: C₆₀H₉₂N₈O₁₁
Purity: ≥ 98.0%
Appearance of Solid: White powder
Solubility: Freely soluble in ethanol
Precautions
1. Unopened filter-sterilized Aureobasidin A solution can be stored stably at -20°C for 2 years. Discontinue use if contamination is found after opening.
2. This product is for research use only by qualified professionals and must not be applied to clinical diagnosis or medical treatment.
3. Wear a lab coat and disposable gloves during operation for personal safety and health protection.
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| 批号(Lot Number) | 证书类型 | 货号 |
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| 分析证书 | A1524600 |
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