Mastercycler nexus gradient

The DNA of the plasmids was isolated using a GeneMATRIX Plasmid Miniprep DNA Purification Kit (EURx, Gdansk, Poland) or the classical alkaline lysis procedure [30 (link)]. Routine DNA manipulations were carried out using standard molecular biology methods [31 ]. DNA was amplified by PCR using a KAPA HiFi PCR Kit and appropriate primer pairs (Table S1). DNA amplification was performed using a Mastercycler nexus gradient, model number F283971R (Eppendorf, Hamburg, Germany). Each thermocycle started with an initial denaturation at 95 °C for 3 min followed by 30 cycles of denaturation at 98 °C for 20 s, annealing at 56 to 63 °C (depending on the primer pair) for 15 s, extension at 72 °C for 1 min/kb, and finished with a final extension at 72 °C for 1 min/kb. The PCR-amplified DNA fragments were then cloned in the pABW1 or pBBR1MCS-2 vectors.
Plasmids constructed in this study were introduced into Achromobacter sp. LM16R, A. tumefaciens LBA288, P. aeruginosa PAO1161 and V. paradoxus EPS by triparental mating [32 (link)], and into E. coli BR825 by chemical transformation [33 ,34 (link)].

SAOS-2 cells were seeded on surfaces, which were not BSA blocked, at a concentration of 1 × 10⁶ cells/cm², and incubated in growth media at 37 °C in 5% CO₂ until confluence. At confluence, the media in selected samples were augmented with osteogenic supplements for the duration of the assay.
RNA was extracted from all samples at 1, 6, and 10 days post-confluence using a High Pure RNA Isolation kit (Roche). The RNA yield and purity were determined using a NanoDrop 2000c (Thermo Scientific). Complementary DNA (cDNA) synthesis was then performed using a Transcriptor First Strand cDNA Synthesis Kit (Roche). Samples were heated to 25 °C for 10 min, 55 °C for 30 min, and 85 °C for 5 min in a Mastercycler Nexus Gradient (Eppendorf). Real-time qPCR was then performed to investigate the change in transcript expression of collagen 1 (COL1), osteocalcin (OCN), osteopontin (OPN), and ALP, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference using a Lightcycler 480 II instrument (Roche). Forward and reverse primers were supplied by Sigma Aldrich (GAPDH, COL1, OCN, and ALP), using sequences described by Liskova et al.,⁴⁷ (link) shown in Table I, and Biorad (OPN). Transcript levels were presented as a fold increase over the day 1 post-confluence expression levels on the untreated bare PEEK surface.

Total RNA was isolated using the RNeasy Mini-Kit (Qiagen). For cDNA synthesis, 200 ng of total RNA was used with the Affinity Script multi temperature cDNA synthesis kit (Agilent). Cdk6 primers were 5′-ACTTGAAGAACGGAGGCCGTTT-3′ and 5′-AAGG CCGAAGTCAGCGAGTTTT-3′. β2-microglobulin was used as a control (5′-GATGCTGCTTACATGTCTCG-3′ and 5′-CCAGCAGAGAATGGAAAGTC-3′). PCR amplifications were performed on a Mastercycler Nexus gradient (Eppendorf).