Failsafe pcr system

Standard PCR was performed using the FailSafe PCR System (EpiCentre, Chichago, IL, USA) with the following amplification conditions: 25 cycles of 50 s at 95 °C, 50s at 60 °C and 1 min at 72 °C. The primers designed (Supplementary Table 2) for variant detection are common forward primer targeted the exon 1, and two distinct reverse primers targeted exon 5 and exon 7, respectively.

Cloning was performed using standard molecular biology techniques. PCR was performed with FailSafe PCR System (Epicentre) using the C. sporogenes genomic DNA as a template with primers PM003 (5′-GGATGTACTACAAGTGCTATTCAC-3′) and PM008 (5′-AGTGCTGGATTTAGTCTAC-3′). The PCR product was cloned into pJET1.2 (Thermo Fisher Scientific) and sequenced. For protein overexpression, the fldZ gene was codon optimised for expression in E. coli with GENEius software (Eurofins MWG Operon) and synthesised with NdeI (at the 5′-end) and NotI (at the 3’-end) restriction sites. After restriction digestion with NdeI and NotI (Thermo Fisher Scientic) the insert from the cloning vector was ligated into pET20b(+) cut with the same restriction enzymes, to form the plasmid pET20b(+):CsfldZ. Cloning of other expression plasmids is described in the Supplementary information 3.

C. acetobutylicum ATCC 824 and C. sporogenes ATCC 15579 genomic DNAs for use in cloning and PCR analysis were prepared using QIAGEN DNeasy Blood and Tissue Kit in accordance with the manufacturer’s instructions and recommended pretreatment for Gram-positive bacteria using Lysozyme from chicken egg white (Cat L6876 Sigma-Aldrich) 20 ml/ml phosphate buffer saline (PBS) [25 (link)]. Screening PCRs were performed using DreamTaq™ green PCR master mix (Fisher Scientific UK Ltd) and Failsafe™ PCR system (Epicentre) for downstream cloning in accordance with the manufacturer’s instructions. Primers were designed using web tool available at http://primer3.ut.ee and are listed in Additional file 1: Table S1. All the DNA manipulations including restriction digestion, de-phosphorylation of 5′ end and ligation were carried out according to standard procedures as described in [44 ]. DNA extraction and purification from agarose gel and PCR reaction mixtures were undertaken using QIAquick Gel Extraction and PCR Purification Kit (Qiagen Ltd UK), respectively, and used according to the manufacturer’s instructions.