Skip to main content
Fig. 1 | Biotechnology for Biofuels

Fig. 1

From: Metabolic engineering of Yarrowia lipolytica for thermoresistance and enhanced erythritol productivity

Fig. 1

The schematic flow to obtain authentic URA3 disruption mutant. The 1.2 kb upstream (lane 1, a) and downstream (lane 2, a) of the ura3 gene was amplified separately, then the 2.4-kb fragment (lane 3, a) was obtained by fusion PCR to merge 1.2 kb up and downstream. Then the 2.4-kb fragment was transformed into HCY109 (ery929Ku70), plated on YPDF medium (10 g/L yeast extract, 5 g/L tryptone, 10 g/L glucose, 2 mg/ml 5-FOA, 15 g/L agar powder, pH 6.5) and cultivated at 30 °C for 5 days (b). Colonies were grown (c) and transferred to a new YPDF plate cultivated at 30 °C for 5 days (d). Then the colonies were transferred to synthetic complex medium without uracil (SC-U, 10 g/L yeast nitrogen base, 5 g/L ammonia sulfate, 10 g/L glucose, without uracil) (e). If the ura3 gene was authentically disrupted, the mutants cannot grow on the SC-U medium (1, 2, 3 in e), or they are not authentic ura3 gene disruption (4, 5, 6 in e). Then two putative ura3-deficient mutants were streaked on SC-U plate (f) and YPDF plate (g), they showed opposite growth. A ura3-deficient mutant was further to be identified by PCR to amplify the 480-bp fragment in ura3 gene, and no 480-bp fragment can be amplified (lane 4 in h), but the 480-bp fragment can be amplified for the control strain (HCY109, ery929Ku70) (lane 5 in h). The No. 1 colony in g designated as HCY109-2 was used as the host to conduct metabolic engineering

Back to article page