Supplementary Materials
Cloning and Characterization of Indole Synthase (INS) and a Putative Tryptophan Synthase a-Subunit (TSA) Genes from Polygonum tinctorium
Zhehao Jin1·Jin-Hee Kim1·Sang Un Park2·Soo-Un Kim1,3
* S.U. Kim
1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea
2 Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-Gu, Daejeon 34134, Republic of Korea
3 College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434023, China
Table S1 Primers used in this study.································································2
Fig. S1 Indole-related enzymes in P. tinctorium and E. coli. ··································3
Fig. S2 Alignment of IGL and IGL-like amino acid sequences to design primer pair for IGL cloning.····················································································4
Fig. S3 Deduced evolutionary course of PtINS from ancestral PtTSA. ····················5
Fig. S4 Strategy for construction of E. coli FS1576 knock-out mutants using homologous recombination.····································································6
Fig. S5 Confirmation of the double knock out E. coli mutants by PCR and indole test. ·········································································································7
Fig. S6 Growth of the double mutant E.coli ΔtrpA ΔtnaA on M9CG plate with (right) or without indole (left). ···············································································8
Fig. S7 Growth of E. coli ΔtrpA ΔtnaA harboring pMW118-PtIGL-longD42 on M9CG plate without indole. ··············································································9
Preparation of knock-out E. coli. ································································10
Supplement Table S1 Primers used in this study. Standard IUPAC-mixed base designations are used with Y(C,T), R(A,G), K(G,T), H(A,T,C), V(G,A,C), and N(G,A,T,C). Underlined ATG denotes translation start site.
Pt-F / ATYGARYTRGGNGTRCCH
Pt-R / RATYACVCCRTCHGCKCCCCA
GeneRacer 5` primer / CGACTGGAGCACGAGGACAGA
GeneRacer 3` primer / GCTGTCAACGATACGCTACGTAACG
Pt5` RACE / TGCCACCTGTGCTACGTGCTCCGGT
Pt3` RACE / AAGGGAGGCTGGTGTACATGGACT
long 5UTR F / CAAACATCACCCCATTTTCCCCAATT
long 5UTR R / CGAGCGAGGCCATGATCTTGGT
short 5UTR F / AGCCCAGCTCCGTCGTCGCCT
short 5UTR R / GCGAGAGAGGCCATGATCTTGGT
PtIGL L-F / ATGGCTGTCTCCCTCAGATCA
PtIGL S-F / ATGGCCTCTCTCGCGACTTCGA
PtIGL-R / TCAAACAAGGGCAGATTTCAGGGATT
pMW118:Pt L-F / GGTACCCGGGGATCTATGGCTGTCTCCCTCAGAT
pMW118:Pt S-F / GGTACCCGGGGATCGATGGCCTCTCTCGCGACTT
Δ42-F / GGTACCCGGGGATCGATGGCCTCGCTCGCTACTTC
pMW118:Pt-R / GACTCTAGAGGATCCTCAAACAAGGGCAGATTTC
pMW118:AtIGL-F / GGTACCCGGGGATCGATGGATCTTCTCAAGACTC
pMW118:AtIGL-R / GACTCTAGAGGATCCTCAAGAGACAAGAGCAGA
pMW118:AtTSA-F / GGTACCCGGGGATCGATGGCGATTGCTTTCAAAT
pMW118:AtTSA-R / CGACTCTAGAGGATCCTCAAGAAGAGCAGATTA
gtnaA-F / TCTGGCGAATTAATCGGTATAGCAG
gtnaA-R / ATGATGCCACCTTTAGAGGAAGGC
Kan-F / CACGTAGAAAGCCAGTCCGCAG
Kan-R / ATAGGGGTTCCGCGCACATT
gtrpA-F / GCCCTGCTCGGCCTGAAAT
gtrpA-R / CAGGTTCGATTCGGACTCCA
chl-F / ATCGATTGTGACGGAAGATCACTT
chl-R / ATCGATACCAGCAATAGACATAAG
Pt LR-F / CTTTGTTTTCCCTAATTCGGCAA
Pt LR-R / CGGCGCTGGAGAAAGCAAGT
Pt SR-F / ATGGCCTCTCTCGCGACTTC
Pt SR-R / CCACAAGTGTCAAGCAACTTCAA
pEAQ:GFP L-F / GCCCAATTCGCGACCATGGCTGTCTCCCTCAGAT
pEAQ:GFP S-F / GCCCAATTCGCGACCATGGCCTCTCTCGCGACT
pEAQ:GFP-R / CTCCTTTAGTCATACCAACAAGGGCAGATTTCAG
Supplement Fig. S1 Indole-related enzymes in P. tinctorium and E. coli. IGP, indole-3-glycerol phosphate; BX1, benzoxazinless 1; TS complex, tryptophan synthase complex; INS (or IGL), indole synthase (or IGP lyase); tnaA, tryptophanase
Supplement Fig. S2 Alignment of IGL and IGL-like amino acid sequences to design primer pair for IGL cloning. Arrows indicate the regions corresponding to primer sequences, Pt-F and Pt-R (Supplement Table S1). AtIGL, AAP04082; At TSA , AAM65526; CoIGL, ACJ02769; ItTSA, CAH56478; LgIGL, ACJ02773; OsIGL, AK073440; OtTSA, CAL51431; ZmTSA, ACA25187.
Supplement Fig. S3 Deduced evolutionary course of PtINS from ancestral PtTSA. Introns in coding region was not determined.
Supplement Fig. S4 Strategy for construction of E. coli FS1576 knock-out mutants using homologous recombination. R gene, antibiotics resistance gene.
Supplement Fig. S5 Confirmation of the double knock out E. coli mutant by PCR and indole test. (A) Electropherogram of PCR products of E. coli FS1576 wild type and DtnaADtrpA mutant with primer pair gtnaA-F and gtnaA-R, respectively. (B) Same as A except PCR with primer pair gtrpA-F and gtrpA-R. Lane M indicates DNA size marker. (C) The double mutant did not produce indole as indicated by lack of reaction color with Kovac’s reagent.
Supplement Fig. S6 Growth of the double mutant E.coli ΔtrpA ΔtnaA on M9CG plate with (right) or without indole (left).
Supplement Fig. S7 Growth of E.coli ΔtrpA ΔtnaA harboring pMW118-PtIGL-longD42 on M9CG plate without indole.
Preparation of knock-out E. coli
To disable tnaA and trpA in E. coli, insertional knock-out strategy (Supplement Fig. 4) was employed by inserting a kanamycin-resistance conferring aphII inside tnaA and chloramphenicol-resistant cfr into trpA (Supplement Fig. 4). An 1.4-kb tnaA ORF fragment, named gtnaA was amplified from E. coli FS1576 (Yin et al. 2010) chromosomal DNA through PCR by using gtnaA-F and gtnaA-R primer pair. The PCR product was cloned into pUC19 vector (Clontech, USA) to construct gtnaA-pUC19, which was then digested with NruI restriction enzyme. The Kan-F and Kan-R amplified aphII was ligated to NruI-digested gtnaA-pUC19 recombinant plasmid, resulting in gtnaA-aphII. The 2.7-kb-long gtnaA-aphII fragment was isolated by PCR by using gtnaA-F and gtnaA-R primer pair and transformed into E. coli FS1576 strain by heat-shock method. The transformant was selected on LB plate containing 50 μg/μl kanamycin to give insertion knock-out mutant, E. coli DtnaA. The mutant was confirmed by PCR using primer pair of gtnaA-F and gtnaA-R.
Next, 1.4-kb-long trpA ORF fragment, named gtrpA, was amplified from E. coli FS1576 chromosomal DNA through PCR by using gtrpA-F and gtrpA-R primer pair. The PCR product was cloned into pUC19 (Clontech, USA) to construct gtrpA-pUC19, which was then digested with ClaI. Chloramphenicol resistance gene (cfr) of 1.1-kb was cloned from pACYCDuet-1 vector (Novagen, Germany) by using chl-F and chl-R primer pair. The gene cfr was ligated to ClaI-digested gtrpA-pUC19 recombinant plasmid, resulting in gtrpA-cfr. The 3.1-kb-long gtrpA-cfr fragment was isolated by PCR by using gtrpA-F and gtrpA-R primer pair and transformed into E. coli FS1576 ΔtnaA strain by heat-shock method. The transformant was selected on LB plate containing 50 μg/μl of kanamycin and 34 μg/μl chloramphenicol and confirmed by PCR using primer pair gtrpA-F and gtrpA-R. The double mutant was named E.coli ΔtnaA ΔtrpA.
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