Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006 Supporting Information for Expanding the Genetic Code in a Mammalian Cell by the Introduction of 4-Base Codon/Anticodon Pairs Masumi Taki, Jiro Matsushita and Masahiko Sisido* Experimental Section 1. Construction of plasmids that express EGFPs 1.1. Plasmid for wild-type EGFP: An NheI restriction site, a Kozak sequence for translation initiation with an ATG start codon, and a FLAG-tag were inserted by PCR just before the initiation codon of the EGFP-encoding gene that originated from pegfp-n3 (Clontech). A HindIII restriction site was also added just after the stop codon of the gene. By using the NheI and HindIII sites, the amplified gene was subcloned into a vector (pcdna5/frt; Invitrogen, Carlsbad, CA) to generate a plasmid that expresses wild-type EGFP (pcdna5/ FRT/EGFPwt) Plasmids for amber mutated EGFP: First, the 66 TAC codon of wild-type EGFP was replaced by an amber codon "TAG" using an improved site-directed mutagenesis based on a QuikChange TM protocol. The sequences of the sense and antisense primers were, 5'- GAC CAC CCT GAC CTA GGG CGT GCA GTG C-3' and 5'-GCA CTG CAC GCC CTA GGT CAG GGT GGT C-3', respectively, with the mutated positions being under-lined. Note 1

3 that site-directed mutagenesis was successfully carried out simply by using the primer pair whose melting temperature on both sides of the mutation position is estimated to be C, from the contents of G:C (+4 C) and A:T (+2 C) pairs [B. Cormack, Current Protocols in Molecular Biology, Wiley, New York, 1991, ]. These primers were purchased from Hokkaido System Science Co., Ltd. (Sapporo, Japan). The site-directed mutation was successful with the primers that were not puri-fied by HPLC, but purified simply and inexpensively by using a reverse-phase cartridge. The 50 µl-scale PCR was carried out with 250 ng of the template DNA (pcdna5/frt/egfpwt), 0.3 µm of the primer pair, 500 µm of dntps and 2.5 U of Pfu Turbo DNA polymerase (Stratagene). The PCR was initiated by preheating the reaction mixture at 95 C for 1 min, followed by 16 cycles of 94 C for 30 s, 55 C for 1 min and 68 C for 12 min. The resulting PCR mixture was treated with a restriction enzyme DpnI (New England Biolabs) for 1 h without further purification in the same PCR tube, and an aliquot (1 µl) of the mixture was directly transformed into DH5α chemocompetent cells. The transformed cells were cultivated on a Luria-Bertani (LB) plate containing ampicillin. The colonies were selected and their plasmids were isolated by a Plasmid Miniprep Kit (Bio-Rad), to obtain the mutated plasmid (pcdna5/frt/ EGFP 66TAG ) Plasmids for EGFPs that contain 66 UAGN, 66 CNCU, 66 CUAU and 66 UUGU 4-base codons, and that contain 66 UAGNN 5-base codons: The plasmid for the EGFP that contain 66 UAGN 4-base codons was prepared by essentially the same procedure as described above, except for using pcdna5/frt/egfp 66TAG as the template plasmid and using the following mixed oligonucleotides as sense primers, 5'-GAC CAC CCT GAC CTA GNG GCG TGC AGT GC-3'; and as antisense primers, 5'-GCA CTG CAC GCC NCT AGG TCA GGG TGG TC-3'. A total of 12 colonies were selected, and their plasmids were isolated by a Plasmid Miniprep Kit and sequenced. All four mutants that con-tain one of the TAGN codons were obtained through this one-step transformation, i.e., four independent EGFP-expression vectors containing the four different types of 4-base codons (pcdna5/ FRT/EGFP 66TAGN ) were simultaneously generated in a single PCR tube from a single template vector that contains an amber codon (pcdna5/frt/egfp 66TAG ). The plasmids for the EGFP that contain 66 CNCU, 66 CUAU, 66 UUGU, and 66 UAGNN codons, were also prepared by essentially the same procedure as above, except for using the following oligonucleotides: 5'-GAC CAC CCT GAC CCN CTG GCG TGC AGT GC-3' 2

4 and 5'-GCA CTG CAC GCC AGN GGG TCA GGG TGG TC-3' for 66 CNCU; 5'-GAC CAC CCT GAC CCT ATG GCG TGC AGT GC-3' and 5'-GCA CTG CAC GCC ATA GGG TCA GGG TGG TC-3' for 66 CUAU; 5'-GAC CAC CCT GAC CTT GTG GCG TGC AGT GC-3' and 5'-GCA CTG CAC GCC ACA AGG TCA GGG TGG TC-3' for 66 UUGU; and 5'-CAC CCT GAC CTA GNN GGC GTG CAG TGC-3' and 5'-GCA CTG CAC GCC NNC TAG GTC AGG GTG GTC-3' for 66 UAGNN. 2. Construction of trna-expressing plasmids 2.1. Plasmid for trna that contains an amber anticodon: A pgem-t-tr-hutyr(cta) plasmid was designed to encode 5 - and 3 -flanking sequences derived from the human trna Tyr gene and a trna-expressing sequence containing an internal promoter and an amber anticodon. The following oligonucleotides were annealed: forward, 5'-GGT TTT TCT GTG CTG AAC CTC AGG GGA CGC CGA CAC ACG TAC ACG TCC CTT CGA TAG CTC AGC TGG TAG AGC-3'; reverse, 5'-GCA CTT GTC TCC TTC GAG CCG GAA TCG AAC CAG CGA CCT AAG GAT CTA GAG TCC TCC GCT CTA CCA GCT GAG C-3'. The primer extension reaction was performed by using a thermal cycler with Ex Taq TM DNA polymerase (Takara, Shiga, Japan). By using this reaction product as the template DNA, PCR was carried out with a sense primer, 5'-GGC TCT AGA ACC TGC CGG CCA CCA GCG CTC CGG TTT TTC TGT GCT GAA CC' and an antisense primer, 5'-CTG CCC GAC CTG CTC ACG CAT GAA AAA AAC CGC ACT TGT CTC CTT CGA G-3'. The PCR product was cloned into pgem-t Easy vector (Promega) according to the manufacturer s protocol to obtain pgem- T-tR-HuTyr(cta) Plasmids for trnas that contain 4-base anticodons (ncua, agng, auag, and acaa), and 5-base anticodons (cccua): The plasmids for trnas that contain one of the 4-base anticodons, ncua, were prepared by essentially the same procedure as described above, except for using pgem-t-tr-hutyr(cta) as a template plasmid and the following oligonucleotides as a sense primer, 5'-GAC CTA AGG ATC TAG NAG TCC TCC GCT CTA C- 3'; and as an antisense primer, 5'-GTA GAG CGG AGG ACT NCT AGA TCC TTA GGT C- 3'. The colonies were selected, their plasmids were isolated by Plasmid Miniprep Kit and sequenced. All four mutants of pgem-t-tr-hutyr(ncta) that contained ncta anticodons were obtained through this one-step transformation. The plasmids for trnas that contain 4-base anticodons agng, auag, acaa, and 5-base 3

5 anticodon cccua were prepared by essentially the same procedure as described above, except for using the following oligonucleotides, 5'-GCG ACC TAA GGA TCC NCT AGT CCT CCG CTC TA-3' and 5'-TAG AGC GGA GGA CTA GNG GAT CCT TAG GTC GC-3' for agng; 5'-GCG ACC TAA GGA TCC TAT AGT CCT CCG CTC TAC-3' and 5'-GTA GAG CGG AGG ACT ATA GGA TCC TTA GGT CGC-3' for auag; 5'-GCG ACC TAA GGA TCT TGT AGT CCT CCG CTC TAC-3' and 5'-GTA GAG CGG AGG ACT ACA AGA TCC TTA GGT CGC-3' for acaa; 5'-GAC CTA AGG ATC TAG GGA GTC CTC CGC TCT-3' and 5'- AGA GCG GAG GAC TCC CTA GAT CCT TAG GTC-3' for cccua. 4

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