Computational protocol: Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

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[…] The strains used in this study are listed in . DNA cassettes were integrated to express GFP-fused FIG1 as follow: DNA fragments containing FIG1(50 bp)-GFP-URA3-TFIG1 (TFIG1: FIG1 terminator) were amplified from pBlue-UFt-AcGFP1, pBlue-UFt-TagGFP2, pBlue-UFt-mUkG1, pBlue-UFt-ZsGreen, pBlue-UFt-yEGFP, pBlue-UFt-yAcGFP1, pBlue-UFt-yTagGFP2, pBlue-UFt-ymUkG1 and pBlue-UFt-yZsGreen using the primer pair 57 and 58. BY4741 was transformed with the amplified DNA fragments using the lithium acetate method. The transformants were selected on SD-Ura plates (SD solid medium without uracil, but containing leucine, histidine and methionine). After confirming the correct integration, the URA3 marker was “popped-out” by homologous recombination using counter-selection with 5-fluoroorotic acid (5-FOA, Fluorochem, Derbyshire, UK), to yield BYFAG1, BYFTG1, BYFUG1, BYFZG1, BYFEG2, BYFAG2, BYFTG2, BYFUG2 and BYFZG2.The STE18 gene was substituted by kanMX4 in the yeast chromosome by amplifying the DNA fragment containing PSTE18–kanMX4–TSTE18 (PSTE18: STE18 promoter and TSTE18: STE18 terminator) from pGK426-GPTK using the primer pair 45 and 46. BYFUG2 was then transformed with the amplified DNA fragment, and transformants were selected on YPD solid medium containing G418 (500 ng/mL) (Nacalai Tesque, Kyoto, Japan) to yield the UGW2 strain.DNA cassettes for the Gγcyto–Fc protein in the cytosol were integrated as follows. DNA fragments containing URA3-PPGK1-Gγcyto-Fc-TPGK1-THIS3 (THIS3: HIS3 terminator) were amplified from pUMGP-GγMFcH using the primer pair 61 (containing the homologous regions of the HIS3 promoter) and 62. UGW2 was transformed with the amplified DNA fragments using the lithium acetate method. The transformants were selected on SD-Ura plates (containing leucine, histidine and methionine) to yield UGFG2.DNA cassettes used to express the membrane-associated Fc protein were integrated as follows: DNA fragments containing PSTE18-PPGK1-Fc-Ste18C-TPGK1-kanMX4-TSTE18 and PSTE18-PPGK1-Gpa1N-Fc-TPGK1-kanMX4-TSTE18 were amplified from pUMGPTK-Fc-Ste18C and pUMGPTK-Gpa1N-Fc, respectively, using the primer pair 59 and 60. BYFUG2 was transformed with the amplified DNA fragments using the lithium acetate method. The transformants were selected on YPD solid medium containing G418 (500 ng/mL) to yield BYFUG2-FC and BYFUG2-FN. DNA cassettes for the Gγcyto–Z domain variants (ZWT, ZK35A, ZI31A and Z955) in the cytosol were integrated as follows: DNA fragments containing URA3-PPGK1-Gγcyto-ZWT(-ZK35A, -ZI31A and -Z955)-TPGK1-THIS3 and URA3-PPGK1-Gγcyto-TPGK1-THIS3 were amplified from pUSTE18p-Gγcyto-ZWT(-ZK35A, -ZI31A and -Z955)-HIS3t and pUSTE18p-Gγcyto-HIS3t using the primers 61 (containing the homologous regions of the HIS3 promoter) and 62. BYFUG2-FC and BYFUG2-FN were transformed with the amplified DNA fragments using the lithium acetate method. The transformants were selected on SD-Ura plates containing leucine, histidine and methionine to yield UG2-FCGW, UG2-FCGK, UG2-FCGI, UG2-FCG9, UG2-FCG0, UG2-FNGW, UG2-FCGK, UG2-FCGI, UG2-FCG9 and UG2-FCG0.The constructed strains were used for the assays or plasmids were introduced using the lithium acetate method. All strains and transformants used for the assays are listed in . [...] Flow cytometry analysis was conducted as described previously. The harvested cells were diluted into test tubes containing the sheath solution, and GFP fluorescence was measured by using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). The green fluorescence signal from 10,000 cells was excited with a 488-nm blue laser and collected through a 530/30-nm band-pass (GFP) filter. The data were analyzed by using BD FACSDiva software (version 5.0, BD Biosciences) and FlowJo software version 7.2.2 (Treestar, Inc., San Carlos, CA). […]

Pipeline specifications

Software tools ggCyto, BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Saccharomyces cerevisiae, Homo sapiens