Subsequently, two independent ethanol tolerant C. thermocellum mutants named E50A and E50C were selected using a strategy that alternated between increasing ethanol concentrations and transfers in medium that lacked selection pressure [ 22].
The co-transformants were selected on synthetic complete selection medium lacking Leu, Trp and His.
Haploid strains containing a 415 bp duplication of a portion of the HIS3 coding sequence flanking the plasmid YIp5 [80] that had been inserted into the HIS3 locus [75] were streaked to single colonies on medium lacking uracil to maintain selection for the URA3 marker on the plasmid.
Stable transformants were obtained by at least two rounds of single spore isolation or three rounds of transfers to selection medium lacking uridine in case of non-sporulating mutants.
At the same time, stable integration rate (or rate of establishment of stable episomal maintenance) of the p1.1eGFP plasmid was 24 times higher than that of the p1.1 EBVTR- eGFP control p1.1 EBVTR- eGFPelecontroledium lacking both HT and MTX (Table 2), clearly indicating that the EBVTR element was active in the very large explasmidn plasmin.
Primary selection was carried out on selective medium lacking the amino acids, histidine and adenine, and secondary selection was carried out using a semiquantitative color assay for β-galactosidase.
Medium lacking serum was termed 'basal medium' (BM), while medium containing 10% FBS was termed as "full medium" (FM).
Positive clones were multiple re-streaked on medium and high stringency selection media (SD medium lacking Trp, Leu, His and Ade, supplemented with 2.5 mM 3-AT and 4 mg/ml X-α-gal) before purification and sequencing of the harboured plasmids.
Synthetic complete medium lacking uracil was used for the selection of URA+ strains.
After 2 d of growth at 30°C, the yeast was transferred to four different selection plates containing SD medium lacking Leu, Trp, and Ade (-LTA) and SD lacking Leu, Trp, and His (-LTH), supplemented with 5, 10, or 15 mM 3-amino-1,2,4-triazole (3-AT), respectively.
