UTK Knoxville- September

9/8/12
Site-directed mutagenesis performed on pLNCX2 vector to correct Neomycin restriction site that conflicts with BioBrick construction.

9/9/12
Colonies picked from plates of Neomycin site-directed mutagenesis

9/10/12
Neomycin site-directed mutagenesis cultures were plasmid extracted and check digestions performed to confirm corrected digestion site. Correct plasmids were then used as template for Neomycin PCR.

9/11/12
Digestions performed:
J63010 + pIRES + GFP + cyc: Xba and PstI
J63010 +pIRES2 + GFP + cyc: Xba and PstI
J63010 +pSAP + GFP + cyc: Xba and PstI
J63010 +YAP1 + GFP + cyc: Xba and PstI
J63010 +URE2 + GFP + cyc: Xba and PstI
J63010 + HAP4 + GFP + cyc: Xba and PstI
J63010 +Adh + mOrange : SpeI and PstI

Ligations performed to create constructs:
J63010 +Adh + mOrange + pIRES + GFP + cyc
J63010 +Adh + mOrange + pIRES2 + GFP + cyc
J63010 +Adh + mOrange + pSAP + GFP + cyc
J63010 +Adh + mOrange + YAP1 + GFP + cyc
J63010 +Adh + mOrange + URE2 + GFP + cyc
J63010 +Adh + mOrange + HAP4 + GFP + cyc

9/21/12
Colony PCR Confirmed:
J63010 +Adh + mOrange + pIRES + GFP + cyc
J63010 +Adh + mOrange + pIRES2 + GFP + cyc
J63010 +Adh + mOrange + pSAP + GFP + cyc

9/23/12
Colony PCR Confirmed:
J63010 + Adh + Neo
J63010 +Adh + mOrange + YAP1 + GFP + cyc
J63010 +Adh + mOrange + URE2 + GFP + cyc
J63010 +Adh + mOrange + HAP4 + GFP + cyc

9/24/12
Digestions:
J63010 + pIRES + GFP + cyc : SpeI and PstI
J63010 + pIRES2 + GFP + cyc : SpeI and PstI
J63010 + pSAP + GFP + cyc : SpeI and PstI
J63010 + Adh + Neo : XbaI and PstI
J63010 +Adh + mOrange + pIRES + GFP + cyc + Adh + Neo : EcoRI and PstI
J63010 + Adh + mOrange +pIRES2 + GFP + cyc + Adh + Neo: EcoRI and PstI
J63010 + Adh + mOrange +pSAP + GFP + cyc + Adh + Neo: EcoRI and PstI
J63010 + Adh + mOrange + cyc : SpeI and PstI
J63010 + Adh + GFP + cyc: SpeI and PstI

9/25/12
Ligations performed to create constructs:
J63010 + Adh + mOrange + cyc + Adh + Neo
J63010 + Adh + GFP + cyc + Adh + Neo
J63010 + pIRES + GFP + cyc + Adh + Neo
J63010 + pIRES2 + GFP + cyc + Adh + Neo
J63010 + pSAP + GFP + cyc + Adh + Neo

9/28/12
Colony PCR Confirmed:
J63010 + Adh + mOrange + cyc + Adh + Neo (AOC)
J63010 + Adh + GFP + cyc + Adh + Neo (AGC)
J63010 + pIRES + GFP + cyc + Adh + Neo (pIRES control)
J63010 + pIRES2 + GFP + cyc + Adh + Neo (pIRES2 control)
J63010 + pSAP + GFP + cyc + Adh + Neo (pSAP control)

BY4741 Yeast electroporated with:
AOC, AGC, pIRES control, pIRES2 control, pSAP control,pIRES complete, pIRES2 complete, pSAP complete.

Electroporations were plated onto 200ug/ml G418 YPD plates.