Computational protocol: Cryptic Polyketide Synthase Genes in Non-Pathogenic Clostridium SPP

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Protocol publication

[…] 27 putative Clostridium KS domains were annotated using SEARCHPKS software and a multiple alignment was performed using ClustalW . Highly conserved regions among Clostridium KS-domain were identified as “VDTMCSS”, “HGTGTSLGD” and “FGGGSN” and these sequences were used to design KS-domain-specific primers. From these degenerated nucleic acid sequences were evolved with a maximum degeneration grade (dg) of 384. Two resulting primer sets were used to amplify DNA encoding KS domains, respectively: pair 1: degKS_1_f: 5′-TKG AYA CWR YNT GYT CAT C-3′ (18 bp, dg 256), degKS_3_r: 5′-TCT CCY AAN GWW GTW CCB GTA CCR TG-3′ (24 bp, dg 384); product size approx. 470 bp and pair 2: degKS_3_f: 5′-CAY GGT ACV GGW ACW WCN TTR GGA GA-3′ (24 bp, dg 384), degKS_5_r: 5′-ATT KGW RCC KCC SRM ACC RAA-3′ (21 bp, dg 256); product size approx. 370 bp. PCR conditions: 100 ng gDNA; 100 pmol primer; program: 94°C 5 min, 94°C 15 sec, 42°C 30 sec, 72°C 1 min, 33×, 72°C 2 min, 4°C hold. The PCR products were cloned into pGEM®T-Easy Vector (Promega Corporation) or pCR®2.1-TOPO® vector (TOPO TA Cloning Kit®, Invitrogen) following the manufacturers protocol, amplified in E. coli and sequenced by Eurofins MWG. The resulting sequences have been deposited in GenBank (Accession No. HE586558 - HE586565) and were analyzed using BLASTX software (http://blast.ncbi.nlm.nih.gov/Blast.cgi) Significant similarities (>75%) to published sequences were analyzed. […]

Pipeline specifications

Software tools SBSPKS, Clustal W, BLASTX
Application Drug design
Diseases Citrullinemia