Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

Table 1

Strains and vectors used in this study.

Strain or plasmid	Description^a,b,c	Reference or source
E.coli strains
DH5α	General cloning host.	BRL
ER2925	Host used for cloning experiments involving methylation-sensitive restriction enzymes.	NEB
P. pastoris strain
GS115	Host used for all of the P. pastoris screening and growth experiments.	Invitrogen
Vectors
pIB11	Bacterial RK2-based expression vector harboring Pm/xylS regulatory promoter system with the bla gene as reporter, Km^R.	[6]
pGL3-control  vector	Firefly luciferase reporter vector, Ap^R.	Promega
pBGP1	P. pastoris episomal expression vector harbouring the 𝑃𝐺𝐴𝑃 promoter. E. coli-P. pastoris shuttle vector, Zc^R, Ap^R.	[19]
pPICZ A	P. pastoris expressional vector harbouring the 𝑃𝐴𝑂𝑋1 promoter. E. coli-P. pastoris shuttle vector, Zc^R.	Invitrogen
pPPE3	pBGP1 derivative in which the three BspHI restriction sites are deleted including one situated in the 𝑃𝐺𝐴𝑃 promoter by using site-directed mutagenesis with primer pair 1, 2, and 3, Zc^R, Ap^R.	This study
pPPE8	pPPE3 derivative in which a BspHI restriction enzyme site was inserted in the start of the zeocin-resistance gene by using site-directed mutagenesis with primer pair 4, Ap^R.	This study
pPPE10	pPPE8 derivative in which the zeocin-resistance gene was substituted with the kanamycin-resistance gene from plasmid pIB11. The 𝐾𝑚^𝑅 gene was amplified from pIB11 with primer pair 5 and end digested with BspHI and EcoRV prior to ligation into the corresponding sites of pPPE8, Ap^R, Km^R, G418^R.	This study
pPPE11	pPPE10 derivative in which a NcoI restriction enzyme site was inserted in the start of the Alpha signal by using site-directed mutagenesis with primer pair 6, Ap^R, Km^R, G418^R.	This study
pPPE12	pPPE11 derivative in which the zeocin-resistance gene was inserted as reporter for the modified 𝑃𝐺𝐴𝑃 promoter. The zeocin-resistance gene was amplified from plasmid pBGP1 with primer pair 7 and end digested with NcoI and NotI prior to ligation into the corresponding sites of pPPE11, Ap^R, Km^R, G418^R.	This study
pPPE17	pPPE12 derivative in which the modified 𝑃𝐺𝐴𝑃 promoter was substituted with the 𝑃𝐴𝑂𝑋1. The 𝑃𝐴𝑂𝑋1 promoter was amplified from pPICZ A with primer pair 8 and end digested with SpeI and NcoI prior to ligation into the corresponding sites of pPPE12, Ap^R, Km^R, G418^R.	This study
pIPA1	pPICZ A derivative in which the BspHI restriction enzyme site in the 𝑃𝐴𝑂𝑋1 promoter is deleted by using site directed mutagenesis with primer pair 9, Zc^R.	This study
pIPA4	pIPA1 derivative in which an EcoRI restriction enzyme site is inserted directly upstream of the putative TATA-box in the 𝑃𝐴𝑂𝑋1 promoter by using site-directed mutagenesis with primer pair 10, Zc^R.	This study
pIPA5	pIPA4 derivative in which a NheI restriction enzyme site is inserted downstream of the transcriptional start site in the 𝑃𝐴𝑂𝑋1 promoter by using site-directed mutagenesis with primer pair 11, Zc^R.	This study
pPPE20	Similar to pPPE17 except for that the 𝑃𝐴𝑂𝑋1 promoter was substituted with the modified 𝑃𝐴𝑂𝑋1 promoter from plasmid pIPA5. The 𝑃𝐴𝑂𝑋1 promoter was amplified from plasmid pIPA5 with primer pair 12 and end digested with SpeI and NcoI prior to ligation into the corresponding sites of pPPE12, Ap^R, Km^R, G418^R.	This study
pPPE22	Similar to pPPE17 except for that the 𝑃𝐴𝑂𝑋1 promoter was substituted with the wild-type 𝑃𝐺𝐴𝑃 promoter from plasmid pBGP1 by subcloning the SpeI/MfeI fragment from plasmid pBGP1 into the corresponding sites of plasmid pPPE12, Ap^R, Km^R, G418^R.	This study
pPPE35	Similar to pPPE17 except for that the 𝑍𝑐^𝑅 gene is substituted with the luc+ gene (encoding firefly luciferase). The luc+ gene was amplified from plasmid pGL3-control vector with primer pair 13 and end digested with NcoI and NotI prior to ligation into the corresponding sites of pPPE17, Ap^R, Km^R, G418^R.	This study
pPPE50	Similar to pPPE20 except for that a KpnI restriction enzyme site is inserted in the 𝑃𝐴𝑂𝑋1 promoter about 90 base pairs upstream of the EcoRI restriction enzyme site by using site-directed mutagenesis with primer pair 14, Ap^R, Km^R, G418^R.	This study

 ^aAp: ampicillin; Km: kanamycin; Zc: Zeocin, G418: aminoglycoside antibiotic.
^bSequences of the primer pairs are listed in Table 2.
^cThe target region was subcloned and verified by sequencing for all of the site-directed mutagenesis applications. Also, all cloned regions amplified by PCR were verified by sequencing.