Phusion 2x master mix

PCR products were cloned into pcDNA3.1 (SGA full-env) (47 (link)) or pCR4 TOPO (shortened-env) (Life Technologies) and transformed into One Shot TOP10 competent E.coli or MAX Efficiency Stbl2 competent cells (Life Technologies). Screening for positively transformed E.coli colonies was performed by colony PCR using Phusion 2x Master Mix (Thermo Scientific), universal vector-specific primers M13F/M13R (pCR4 TOPO) or T7/BGHrev (pcDNA3.1), and colonies diluted in 100 μL LB medium (0.5 μL used as template for PCR). LB_Amp cultures of positive clones were grown overnight; plasmids were isolated using the QIAprep Spin Miniprep kit (Qiagen). Plasmids were sequenced for the insert using vector-specific primers. Sequence analysis and assembly were performed using SeqMan Pro (DNASTAR). For subsequent phylogenetic and epitope analysis, all individual sequences per longitudinal env time point and study participant were averaged to consensus (con) sequences using Consensus Maker (Los Alamos National Library (LANL) Database) (www.hiv.lanl.gov) or SeqMan Pro.

We implemented our fully automated protein testing pipeline on an in-house Tecan liquid-handling system and the Strateos Cloud Lab. The system was initialized with a plate of the 34 gene fragments (5 ng μl⁻¹), an NEB Golden Gate Assembly Kit (E1601L) diluted to a 2× stock solution, a 2 μM solution of forward and reverse PCR primers, Phusion 2X Master Mix (ThermoFisher F531L), 2× EvaGreen stock solution, Bioneer AccuRapid Cell Free Protein Expression Kit (Bioneer K-7260) Master Mix diluted in water to 0.66×, AccuRapid E. coli extract with added 40 μM fluorescein, a fluorogenic substrate master mix (139 μM 4-methylumbelliferyl-α-d-glucopyranoside, 0.278% vol/vol dimethylsulfoxide (DMSO), 11 mM phosphate and 56 mM NaCl, pH 7.0) and water.

Quantification of bacterial load was performed to determine by assessing the number of 16S rRNA genes present per gram of faeces using LightCycler® 480 apparatus (Roche), associated with LightCycler® 480 Software (version 1.5; Roche), was used for the real-time PCR. Each reaction contained 5 μl of a 1 in 10 dilution of genomic DNA and was carried out in quadruplicate in a volume of 15 μl in a 384-well LightCycler® 480 PCR plates (Roche), sealed with LightCycler® 480 sealing foil (Roche). Amplification reactions were carried out with Phusion 2X master mix (Thermo Fisher Scientific) using run conditions, primers (0.5 pmol each per reaction) and probe (0.1 pmol per reaction) as described by Furet et al. [55 (link)]. Quantitation was done by using standard curves made from known concentrations of linearized plasmid DNA containing the 16S rRNA amplicon. Wells containing nuclease-free water were included as negative controls. Statistical analysis was performed using Graphpad Prism 5, whereby a one-way ANOVA followed by the Tukey test determined statistical significance; ***P value < 0.001, **P value < 0.01, *P value < 0.05.