Hindiii

The REGIA collection was used for the screening, as previously described [21 (link)]. Site II element was de novo synthesized with cohesive ends corresponding to HindIII recognition site (Eurofins, Hamburg, Germany). Vector was digested accordingly and the following sequences were inserted in the bait vector by ligation: forward 5’ agc ttT CGT TAG AAA TAT ATT TAA GTA AAG TAT ATT ATG ATA TAT Ac 3’ and reverse 5’ tcg agT ATA TAT CAT AAT ATA CTT TAC TTA AAT ATA TTT CTA ACG Aa 3’. Cohesive ends are in lower case characters. The identity of the prey was confirmed by PCR followed by SANGER sequencing using target specific primers (Supplementary Table S1).

The AFLP analysis was performed as described previously (Vos et al., 1995 (link)), with minor modifications according to Klie et al. (2013) (link). For each sample, 100 ng of DNA was digested with 9 U HindIII (Fisher Scientific – Germany GmbH, Schwerte, D) and 3.5 U MseI (Fisher Scientific - Germany GmbH, Schwerte, D). The preamplification reactions were performed with specific primers that had an A as a selective base at the 3′ end [HindIII (5′-AGACTGCGTACCAGCTT-A-3′) and MseI (5′-GACGATGAGTCCTGAGTAA-A-3′)]. HindIII (5′-AGACTGCGTACCAGCTT-ANN-3′) primers with two extra selective bases and MseI (5′-GACGATGAGTCCTGAGTAA-ANNN-3′) primers with three extra selective bases were used for the final amplification. The HindIII primers were end-labeled with an infrared dye (either IRD 700 or IRD 800; Eurofins MWG, Ebersberg, D). In a single PCR reaction, labeled primers were used either as single primers or in combinations of two differently labeled primers (IRD 700 and IRD 800). In total, 21 selective primer combinations were analyzed (Table 1). The fragments were separated on 6 % polyacrylamide gels (Sequagel XR, Hessle, UK) using a DNA analyzer (LI-COR, Lincoln, Nebraska, USA) and automatically processed using the e-Seq-Software (V3.0, LI-COR, Lincoln, Nebraska, USA).

Both ends of the mIL-1Ra gene encoded by pTAKN-2 were cleaved using the restriction enzymes HindIII and KpnI (TaKaRa Bio, Inc., Tokyo, Japan). The resulting DNA fragment was ligated to pNZ8148#2:SEC digested with HindIII and KpnI. The constructed plasmid (pNZ8148#2:SEC-IL1Ra) was subjected to sequence analysis (Eurofins Genomics) to examine consistency with the desired sequence. pNZ8148#2:SEC-IL1Ra was introduced into L. lactis NZ9000^{15 (link)} to generate the gmLAB (designated NZ-IL1Ra). Simultaneously, the original plasmid lacking the mIL-1Ra gene was also introduced into L. lactis NZ9000 to generate the vector control gmLAB (designated NZ–VC).