Several different plasmids were used as backbone in the design. These contain different yeast selection markers, enabling simultaneous integration and cultivation of yeast. Backbone plasmid p413TEF was used for the CUP based systems. It contains the His3 selection marker for yeast and an ampicillin resistance gene for E. coli cultivation. p415TEF was used for the TetON induction systems, this plasmid contains the Leu2 selection marker for yeast and an ampicillin resistance gene for E. coli cultivation. Backbone plasmid p416TEF was used for the plasmid containing the EstrInd-FatB system, while p416GFP is used for the GFP and EstrInd linked induction system. Both p416TEF and p416GFP contain the Ura3 selection marker and an ampicillin resistance gene.
The last part of the design process before entering the physical lab, involves creating primers containing a 20bp overhang in each end, permitting Gibson assembly of the different ordered gene fragments into plasmids. Primers and thioesterase gene fragments were ordered from IDT and Eurofins. Two of the induction systems, EstrInd and TetON, were kindly provided to us by the Tom Ellis Lab at Imperial College Centre for Synthetic Biology (IC-CSynB) and the Department of Bioengineering at Imperial College, London. All other gene fragments were fortunately provided by the Systems and Synthetic Biology division at Chalmers University of Technology. Our supervisor Andrea, helped us assembly the VP16-Estradiole-zif268 gene fragement for the EstrInd system together with promotor (ScTEF1p) and terminator (ScADH1 T) using MoClo assemblies. The same applies for the TetA-NLS-Gal4AD for the TetON system, which was assembled together with promotor (ScTEF1p) and terminator (ScPGK1 T).
We used PCR amplification for the gene fragments and digested the backbone plasmids using restriction enzymes in preparation for Gibson Assembly Cloning, with one exception. For p416GFP we used PCR-mediated deletion of the non-target sequence to retain the GFP gene in the backbone. In order to verify digestions and successful PCRs we used gel electrophoresis.