Top 10 escherichia coli cells

First, a synthetic gene containing the sequence of the Z domain of Staphylococcus aureus Protein A fused to the CP of TuMV was designed. The Z domain was fused to the N-terminus of the CP as this is the part exposed to the solvent, and both sequences were separated by a flexible linker to avoid steric hindrances (Figure 1). This synthetic gene was designed and ordered from GeneArt (Thermo Fisher Scientific, Regensburg, Germany). This gene construct was cloned in the pEAQ-HT-DEST1 vector [49 (link)] using the top 10 Escherichia coli cells (Thermo Fisher Scientific, Regensburg, Germany) to finally transform cells of Agrobacterium tumefaciens (LBA4404 strains) for transient expression in Nicotiana benthamiana. Transformed bacteria were stored in plates with 50 µg/mL rifampicin and 50 µg/mL kanamycin. An individual colony of transformed A. tumefaciens was subsequently grown for 24 h at 28 °C in LB with 50 µg/mL kanamycin up to an optical density at 600 nm (OD₆₀₀) of 1.2. Cells were pelleted down by centrifugation and resuspended in MMA medium (10 mM MES, pH 5.6, 10 mM MgCl₂ and 150 µM acetosyringone). The culture was then incubated for 2 h at 28 °C. Agroinfiltration in N. benthamiana plants took place using a needle-less syringe, following a protocol described elsewhere [12 (link)].

DEC2 (NM_030762) cDNA was amplified from human primary fibroblast total cDNA and was inserted into pcDNA3.1 V5 hisA vector (Thermo Fisher Scientific). The following primers were used for DEC2 cDNA amplification: sense 5’-AACGAAGGATCCGCCACCATGGACGAAGGAATTCCTCATTTGCA-3’ and antisense 5’-GGACGCCTCGAGTCAGGGAGCTTCCTTTCCTGGCT-3’.
2 kb part of IL-1β promoter (NG_008851.1) was amplified from Human Genomic DNA (Roche Basel, Switzerland; cat# 11691112001) and inserted into pGL3-Enhancer vector (Promega Corporation, Fitchburg, USA). The following primers were used for amplification: sense 5’-AATTTGGGTACCAATGCTGTCAAATTCCCATTCACCCA-3’ and antisense 5’-TACTTCCTCGAGGGCTGCTTCAGACACTTGAGCA-3’. The constructs were validated by using nucleotide sequencing.
For dual-luciferase assay the control vector was pRL-TK (Promega). Vectors were propagated in competent TOP10 Escherichia Coli cells (Thermo Scientific). Ultrapure endotoxin-free plasmid DNA was prepared using NucleoBond^® Xtra Midi EF (Macherey-Nagel, Düren, Germany; cat# 740420) according to the manufacturer’s instructions. Plasmid DNA was diluted in a sterile water.

All promoters were generated to include XhoI and HindIII overhang sequences on the 5′ and 3′ ends, respectively. Several promoters were constructed using ligated oligonucleotides. All sequences for primers and oligonucleotides used in this work are shown in Table 2. PCR-amplified promoters and pNLP3 were digested with XhoI and HindIII, and then promoters were ligated into pNLP3 using T4 DNA ligase (New England BioLabs, Inc.). Ligated constructs were transformed into One Shot electro- or chemically competent TOP10 Escherichia coli cells (number C404052 or number C404010; Thermo Fisher Scientific), which were then plated at several dilutions on LB agar containing ampicillin. Resulting colonies were expanded in LB broth with ampicillin, and plasmid was isolated using the QIAprep Spin miniprep kit (Qiagen). All constructs were confirmed by sequencing.