2.1 Plasmid construction
Plasmids were constructed according to Holkenbrink et al., 2018
[19]. Integration and guide RNA (gRNA) vectors were used to
introduce gene expression constructs into characterized genome sites ofY. lipolytica [19].
Primers, synthetic DNA, BioBricks, and plasmids used in this study are
listed in Supplementary Tables S1, S2, S3, and S4, respectively.
BioBricks were amplified by PCR using Phusion U polymerase (Thermo
Fisher Scientific) with the following thermal program: 98°C for 5 min,
30 cycles of (98°C for 20 s, 54°C for 30 s, 72°C for 30 s/kb), and 72°C
for 4 min. After DNA electrophoresis on 1% agarose gel, BioBricks were
purified using NucleoSpin® Gel and PCR Clean-up kit
(Macherey-Nagel). The integration vectors were digested with FastDigest
SfaAl (Thermo Fisher Scientific) and nicked with Nb.BsmI (New England
BioLabs). Biobricks with compatible overhangs and nicked integration
vectors were assembled and transformed into Escherichia colistrain DH5α via Uracil-Specific Excision Reaction
(USER®) cloning. Clones containing the correct
assembly were verified by PCR and Sanger sequencing.