Product Introduction:

Aureobasidin A (AbA) is A cyclic ester peptide antibiotic isolated from Aureobasidium pullulans No. R106, which has strong antifungal ability. At lower concentrations (0.1-0.5 μg/ml), it is toxic to yeast. Fungal species that are sensitive to it include: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida glabrata, Aspergillus nidulans) and A. niger. The mechanism of action is that AbA inhibits the activity of inositol phosphorylceramide (IPC) synthetase, which is dependent on fungal growth, and interferes with sphingolipid synthesis, thus further killing the strain. The genes encoding IPC synthetase are AUR1 gene from Saccharomyces cerevisiae and AURA gene from Aspergillus nidus, both of which have homology. By mutating these coding genes, such as the AUR1-C gene, strains can become resistant to AbA.

AbA is very suitable as a drug selective marker for screening positive clones. AbA resistance is also an ideal reporter for single/two-hybrid studies in yeast.

Procedure (ABA-resistant yeast conversion system)

1) 0.5ml yeast cultured overnight was added to 50 ml YPD medium (formula: 1L liquid medium contained 10g yeast extract, 20g polypeptone, and 20g D-glucose; Solid medium with additional 2% AGAR;) .

2) Cultured at 30℃ for about 6 hours, OD660 was determined to be 1 ~ 2. When diploid was used, OD660 was determined to be 2-4.

3) Centrifuge 1,000×g for 5 min.

4) Suspend precipitation with 10 ml Solution A (formula: 100 mM Lithium acetate, 10 mM Tris-HCl pH 7.5, 1 mM EDTA) and centrifuge 1,000×g for 5 min.

5) Re-suspension precipitation with Solution A until OD660 is 150.

6) 100 μl cell suspension was separated in the tube and cultured at 30℃ for 1 hour.

7) Add 5 μg Carrier (circular or linear DNA) and 150 μg Carrier DNA (which has been heated at 100 ° C for 10 minutes and cooled rapidly).

Note: pAUR101 requires the use of linear DNA for conversion. Using circular DNA can make the conversion less efficient or even unsuccessful. pAUR112 and pAUR123 require complete plasmid DNA for transformation.

8) Add 850 μl Solution B (Formula: dissolve 40 g Polyethylene Glycol 4000 in 100 ml Solution A to fully dissolve, need to use and mix), gently mix.

9) After 30 minutes at 30℃, culture at 42℃ for 15 minutes.

10) Let stand at room temperature for 10 minutes.

11) Centrifugation at 5,000 rpm for 1 min and suspension precipitation with 5 ml YPD medium.

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

13) Centrifugation at 5,000 to 10,000 rpm and suspension precipitation with 1-10 ml 0.9% NaCl.

14) 100 μl cell suspension was inoculated on a YPD select medium plate containing a certain concentration of AbA, depending on the strain type. The transformation was completed after culture at 30℃ for 3-4 days.

15) Positive inverters were selected and/or conversion efficiency (expressed as the number of colonies transformed per microgram of plasmid DNA) was measured.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.