Transgenic yeast (pAG305GPD-AAE13) vs. control yeast (pAG305GPD-ccdB)

The yeast expression vectors used to express AAE13 was constructed as follows: AAE13 (At3g16170) was amplified from the vector pENTR-AAE13. The amplified product was cloned into pCR®8⁄GW⁄TOPO® (Invitrogen). The resulting plasmid was combined with the Advanced Gateway destination vectors pAG305GPD-ccdB (Addgene, Boston, MA) in an attL X attR recombination reaction to generate the vectors pAG305GPD-AAE13. The integrating vector pAG305GPD-AAE13 was introduced into WAT11, which was also transformed empty vector pAG305GPD-ccdB as control (B1, B2, B3 and B4). The transgenic and control yeast cells were cultured in SD-drop out medium at 30 °C under shaken conditions, malonate was fed into cells at final concentration 0.2 mM when the value of OD600 reached at 0.1. The cells were harvested at mid-log phase. Two-condition experiment, Transgenic yeast (pAG305GPD-AAE13) vs. control yeast (pAG305GPD-ccdB). Biological replicates: 4 control replicates, 4 transgenic replicates.