5 alpha competent e coli

RNA of HeLa-H2B-GFP cells was isolated, cDNA synthesized and sequences of interest (Lamin B1 or LC3B) amplified with PCR using appropriate primers generating an overhang on both ends. Using gel electrophoresis, PCR products were isolated and purification was conducted with gel extraction. With these products, construction of expression vector was performed with linearised pHAGE-CMV-dsRED-vector carrying an ampicillin resistance gene using Gibson Assembly Master Mix (New England Biolabs). Expression vectors were transformed to 5-alpha Competent E. coli (High Efficiency, New England Biolabs) and transformed colonies were picked and cultivated, DNA was isolated and plasmids were sequenced to examine expression vectors. HEK293 cells were transfected with expression vectors, envelope (pMD2.G) and packaging vectors (psPAX2) to produce lentiviruses, which were collected for several days. After that, lentiviruses were filtrated and ultracentrifuged with 20% sucrose. After removing supernatant, viruses were resuspended and used to transduce HeLa-H2B-GFP cells and to generate HeLa-H2B-GFP-Lamin B1-dsRed and HeLa-H2B-GFP-LC3B-dsRed cells.

Escherichia coli strains were purchased from New England Biolabs (NEB), Hitchin, UK; “NEB^® 5-alpha competent E. coli” was used for cloning purposes and “T7 Express competent E. coli” was used for recombinant protein expression. Strains were cultured aerobically in Luria Bertani (LB) broth with shaking or on LB agar (Fisher Bioreagents, Loughborough, UK) at 37 °C unless stated otherwise. Where appropriate, ampicillin was added to a final concentration of 100 µg/mL.
Clostridioides difficile strain 630 (PCR ribotype 012) was kindly provided by Peter Mullany, UCL. Strain R20291ermB was previously generated in which ermB conferring resistance to erythromycin was integrated into the genome of epidemic strain R20291 (PCR ribotype 027) [46 (link)]). Strains were cultured in Brain Heart Infusion medium (Oxoid) supplemented with 5 μg/mL yeast extract and 0.1% w/v L-cysteine (BHIS) containing selective supplements; 250 μg/mL D-cycloserine and 8 μg/mL cefoxitin (Oxoid) (BHIS CC). Strains were incubated overnight at 37 °C in an anaerobic workstation (Don Whitley Scientific, Bingley, UK) with an atmosphere of CO₂ (10%), H₂ (10%) and N₂ (80%).

The sequences of gRNAs used for activating endogenous and exogenous genes are listed in S1 Table. Oligonucleotides of 5’CACC-sense gRNA-3’ and 5’ AAAC-antisense gRNA-3’ were purchased from Integrated DNA Technology (IDT). They were annealed by cooling from 95°C to 25°C for 1.5 hours. Annealing reaction mixtures were prepared as follows: 1μL of sense oligo (100μM), 1μL of antisense oligo (100μM), 10x annealing buffer (400 mM Tris-HCl (pH 8.0); 200 mM MgCl2; 500 mM NaCl) and 7 μL of nuclease free water. The annealed oligonucleotides were cloned into a gRNA expression vector by Golden Gate Assembly.
Golden Gate Assembly reactions were prepared as follows: 2.5 μL of annealed oligo, 1μL of gRNA cloning vector (80ng), 1μL of Esp3I restriction enzyme (NEB, #R0734S), 1.5μL of T4 DNA ligase (NEB, #M0202S), 2 μL 10x T4 DNA ligase buffer and 12.5 μL of nuclease free water. The Golden Gate Assembly reaction was performed in a thermal cycler using the following program: Step 1: 37°C for 5min; Step 2: 16°C for 5min; repeat steps 1–2 for 60 cycles; step 3: 75°C for 5min, step 4: 4°C hold. 5μL of reaction was transformed into NEB 5-alpha Competent E. coli (NEB, #C2987H) by following the manufacturer’s protocol.