Aureobasidin A, also known as AbA, Basifungin, breomycin A, is a cyclic ester peptide antibiotic isolated from the filamentous fungus Aureobasidium Pullulans No. R106. It has a strong anti-fungal ability and is an inhibitor of the inositol phosphorylated ceramide synthase AUR1. It is toxic to yeast even at lower concentrations (0.1-0.5 μg/mL). Species of fungus susceptible to Aureobasidin A include Saccharomyces cerevisiae, Schizosaccharces pombe, Candida glabrata, Aspergillus nidulans and A. niger.Fungal species susceptible to it include dental yeast (Saccharomyces cerevisiae), Schizaccharomyces millet (Schizosaccharomyces pombe), Candida glabrata (Candida glabrata), Aspergillus nest (Aspergillus nidulans) and Aspergillus niger (A. niger.). The mechanism of action is that Aureobasidin A inhibits the activity of inositol phosphoramidite (inositol phosphorylceramide, IPC) synthase, on which fungal growth is dependent, and interferes with sphingolipid synthesis, thus further killing the strain. More genes encoding IPC synthases have been studied as the AUR 1 gene from Saccharomyces cerevisiae and the AURA gene from A. sinensis, which has homology. Muting these coding genes makes the strains resistant to Aureobasidin A, such as the AUR 1-C gene.

Aureobasidin A is highly suitable as a drug selection marker for screening positive clones. Aureobasidin A resistance is also an ideal reporter in yeast single-hybrid and two-hybrid studies. This product is a solution of Aureobasidin A dissolved in methanol with a concentration of 1 mg/mL.

Features

Purity≥97%

Standardized production, using factory mass production mode

Applications

Yeast two-hybrid studies

Yeast one-hybrid studies

Rigorous yeast drug selection marker

Aureobasidin A resistance is an ideal reporter for yeast hybridization studies

Specifications

Components

Shipping and Storage

The Aureobasidin A（AbA） products should be stored at -15℃ ~ -25℃ for 2 years.

Instructions

1. Working concentration   

Please refer to relevant literature for specific concentration, and explore and optimize according to your own experimental conditions (such as experimental purpose, cell type, culture characteristics, etc.)

2. Cell experiment (in vitro experiment)

Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides^[1].

Triple tandem repeats of each CArG-box motif were synthesized. All DNA fragments were cloned into the Y1H vector pAbAi (Clontech, Mountain View, USA) using suitable restriction sites. Then, each assembled pAbAi construct was linearized with BstbI and transformed into Saccharomyces cerevisiae Y1HGold strain according to the Yeast Transformation System 2 Manual (Clontech, Mountain View, USA). Clones carrying the desired DNA fragment were screened for auto activation on synthetic uracil dropout medium supplemented with Aureobasidin A in the concentration of 100–900 ng/mL, as indicated (Clontech, Mountain View, USA)^[2].

The open reading frames (ORFs) of SlBES1 genes were amplified and ligated into pGBKT7-GAL4BD plasmid. The fusion GAL4BD-SlBES1 constructs were further transformed into Y2H Gold yeast cells. The SD/−Trp medium plates were used to cultivate the yeast transformants. The α-galactosidase activity of the transformants was identified by X-α-gal and the expression of AUR1-C was screened by Aureobasidin A^[3].

3. Minimum inhibitory concentrations of Aureobasidin for various yeasts

4. Operating Procedures (for yeast transformation system resistant to AbA)

1) Add 0.5 mL of overnight yeast culture to 50 mL of YPD medium (composition: 1 L liquid medium contains 10 g yeast extract, 20 g polypeptone, 20 g D-glucose; for solid medium, add an additional 2% agar).

2) Incubate at 30℃ for approximately 6 hours, until the OD660 is 1-2. When using diploids, measure the OD660 to be 2-4.

3) Centrifuge at 1,000×g for 5 minutes.

4) Resuspend the pellet in 10 mL of Solution A (composition: 100 mM Lithium acetate, 10 mM Tris-HCl pH 7.5, 1 mM EDTA), then centrifuge at 1,000×g for 5 minutes.

5) Esuspend the pellet in Solution A until the OD660 is 150.

6) Aliquot 100 µL of the cell suspension into a tube and incubate at 30℃ for 1 hour.

7) Add 5 µg of the vector (circular or linear DNA) and 150 µg of Carrier DNA (which has been heated at 100℃ for 10 minutes and then quickly cooled).

Note: pAUR101 requires linear DNA for transformation. Using circular DNA will reduce the transformation efficiency or may even fail. pAUR112 and pAUR123 require complete plasmid DNA for transformation.

8) Add 850 µL of Solution B (composition: dissolve 40 g Polyethylene Glycol 4000 in 100 mL of Solution A and mix well, prepare fresh before use), and gently mix.

9) Incubate at 30℃ for 30 minutes, then at 42℃ for 15 minutes.

10) Place at room temperature for 10 minutes.

11) Centrifuge at 5,000 rpm for 1 minute, and resuspend the pellet in 5 mL of YPD medium.

12) Incubate at 30℃ for 6 hours to overnight.

13) Centrifuge at 5,000-10,000 rpm, and resuspend the pellet in 1-10 mL of 0.9% NaCl.

14) Plate 100 µL of the cell suspension onto YPD selective medium plates (containing a certain concentration of AbA, depending on the strain type). Incubate at 30℃ for 3-4 days until the transformation is complete.

15) Pick positive transformants, and/or determine the transformation efficiency (expressed as the number of colonies transformed per microgram of plasmid DNA).

Documents:

Safety Data Sheet

60231_MSDS_HB220420_EN.pdf

Manuals

60231_Manual_HB250116_EN.pdf

Figures

Figure1. Yeast plate growth chart

Experimental strain:GS115

Usage amount: 0.05 μg/mL、0.1 μg/mL、0.5 μg/mL、1 μg/mL

Treatment: 3-5 Days at 30℃

The upper row is the yeasen product, and the lower row is the brand T *.