Escherichia coli jm109

Based on T-RFLPs results and to detect main shifts in biofilm bacterial composition, selected DNA samples obtained from biofilms grown under steady state conditions on days 7, 14 and 28 were PCR-amplified with bacterial primers 27F (5′-AGAGTTTGATCCTTGGCTCAG-3′) and 1492R (5′-GCYTACCTTGTTACGACTT-3′) (Lane 1991 ). PCR conditions were 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 54°C, and 90 s at 72°C and a final extension for 10 min at 72°C. The PCR master mix was prepared as explained in the previous section. PCR products from three biological replicates were pooled and purified using the QIAquick PCR Purification Kit (Qiagen Inc.). Purified PCR products were cloned using the pGEM-T Easy Vector Systems (Promega UK Ltd, Southampton, UK), and ligations were performed overnight at 4°C. Transformations were carried out using competent cells of Escherichia coli JM109, following manufacturer's instructions (Promega). Transformants were selected by ampicillin resistance, and blue-white screening was performed to identify clones with inserts (Sambrook et al. 1989 ). Inserts were sequenced from both directions using plasmid-vector-specific primers M13F (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and M13R (5′-TAACAATTTCACACAGGA-3′) primers. Samples were purified with EXOSAP and sequenced at The University of Sheffield, Medical School with an Applied Biosystems 3730 automated DNA analyser.

The bacterial strains and plasmids used in this study are listed in Table 1. The L. lactis strains were grown at 30 °C in M17 broth (Oxoid Ltd., Basingstoke, UK) supplemented with 0.5% (w/v) glucose (GM17). Pediococcus damnosus CECT4797 was grown in MRS broth (Oxoid Ltd.) at 30 °C. Escherichia coli JM109 (Promega, Madison, WI, USA) was grown in Luria–Bertani (LB) broth (Oxoid Ltd.) at 30 °C with shaking. Chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA) was added at 20 µg/mL to select growth of E. coli and at 5 µg/mL for the selection of the recombinant lactococcal strains. The cell dry weights of the late exponential phase cultures were determined gravimetrically. Agar plates were made by the addition of 1.5% (w/v) agar (Oxoid) to the liquid media.

The yeast Saccharomyces cerevisiae haploid strains used in this study are listed in Table 4.
The yeast deletion mutants used in this study were isogenic to one of the two parental strains, i.e., W303-1A [70 (link)] or BY4741. The wild type BY4741 strain and its deletion mutants were purchased from the EUROSCARF collection (www.uni-frankfurt.de/fb15/mikro/euroscarf).
Escherichia coli JM109 (endA1, recA1, gyrA96, thi, hsdR17(r_k⁻, m_k⁺)supE44, λ⁻, Δ(lac – proAB), [F', traD36, proAB, lacI^qZM15] strain (Promega) was used for multiplication of plasmids.

Tne strain NS-E (DSM 4359) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). The organism was grown anaerobically in Tma basal medium (TBM) with 0.5% glucose at 80 °C^{14 (link)}.
Escherichia coli JM109 (Promega) was used as host for cloning and expression of genes in a pHsh vector (GenBank accession number: FJ571619.1), constructed by Shine E Biotech, Nanjing, China. E. coli cells were routinely grown aerobically in Luria–Bertani (LB) medium at 30 °C, and 100 μg/ml ampicillin was added to the LB medium for selective cultures.

DNA was isolated from flash-frozen sediments or biofilm, using PowerSoil DNA Isolation Kit (MoBio, Carlsbad, CA, USA) following manufacturer’s instructions. The template DNA was amplified using GoTaq Green Master Mix (Promega, Madison, WI, USA) with addition of 1 µl bovine serum albumin per 50 µl reaction in T100 thermal cycler (Bio-Rad, Hercules, CA, USA). The 16S rRNA gene was amplified using 27F-CM and 1492-R primers [33] (link), yielding ∼1450 bp product; the 23S rRNA gene was amplified with broad specificity forward primer L-0858-a-S-21 and zetaproteobacteria specific L-C-Zeta-1611-A-22 reverse primer [6] (link), yielding ∼750 bp product. The PCR conditions were as follows: denaturation at 94°C for 30 s, annealing at for 30 s 50 °C and extension at 72°C for 1min (1.5 min for 16S) for 35 cycles. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and subsequently cloned in the pGEM-T Easy vector (Promega, Madison, WI, USA) using Escherichia coli JM109 (Promega, Madison, WI, USA) as a host. The sequencing was performed by HyLabs (Israel) using pGEM-T specific primers T7 and SP6.

Escherichia coli JM109 (Promega, Charbonnière, France) was used as a host for the cloning and propagation of vectors. A wild-type A. brasiliensis strain (BRFM103, CIRM-CF, Marseille, France) collected in a temperate forest was used as host for fungal transformation [33 (link)]. The eight recombinant L-LA-producing strains were registered in the CIRM as BRFM1872, BRFM1873, BRFM1874, BRFM1875, BRFM1877, BRFM1879, BRFM1880, BRFM1881.