Procedures with GCase

The glucocerebrosidase (GCase) gene was designed with the amino-terminal Pdi1 peptide targeting sequence and the carboxy-terminal Strep-Tag II. Furthermore, BamHI and PstI restriction sites were added at the 5' and 3' respectively. We amplified this PDI1-GCase-Strep-Tag II gene by PCR and digested the amplicon with BamHI and PstI.

We previously had cloned either the TEF promoter or the GPD promoter (using the restriction enzymes SacI and BamHI), as well as the CYC1 terminator (using the restriction enzyme HindIII), in the integrative shuttle vector YIp352 [1], which confers resistance to ampicillin as a selection mark in E. coli and prototrophy for uracil as a selection mark in S. cerevisiae. Therefore, we cloned the designed glucocerebrosidase with either one of these promoters. We called the plasmids pIUTEFGCase-cyc and pIUGPDGCase-cyc.

After ligation, competent E. coli cells from the RR1 strain were transformed and the ampicillin-resistant colonies were screened by the modified plasmid miniprep protocol followed by digestion with the restriction enzyme EcoRV.

Two ampicillin-resistant colonies were chosen based on the screening and these two had their plasmid DNA extracted by the Triton X-100 lysis procedure. Both plasmids were analyzed by diagnostic digestion as shown.

[1] Hill, J. E., Myers, A. M., Koerner, T. J., & Tzagoloff, A. (1986). Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast, 2(3), 163-167.
We are now finalizing the knockout constructions in Saccharomyces cerevisiae, and we hope to present the new results on this topic in the Judging Session.