Project description:Thermal proteome profiling of E. coli lysate treated with fosfomycin (0.2 mg/ml). Untargeted proteomics with microflow method and with 60 min gradient.
Project description:E.coli ATCC 25922 was overnight grown in 10 ml LB and passaged for 3 h with 1:50 dilution in fresh LB. Treated with tavaborole alone (80 μg/ml, labeled ‘A’) or tavaborole plus amikacin (80 μg/ml +40 μg/ml, labeled ‘AK’) for 6 h, cells were washed in PBS and harvested by centrifugation at 4,000 g, 8 min. After liquid nitrogen flash freezing, cells were stored at -80 °C and transported to company for following experiments
Project description:Genome-wide transcriptional analysis was performed on E. coli K12 exposed to 1 mg/ml of olive vegetation water phenolic extract (OVWPE) in planktonic condition.
Project description:Genome-wide transcriptional analysis was performed on E. coli K12 exposed to 1 mg/ml of olive vegetation water phenolic extract (OVWPE) in planktonic condition. Three groups of samples were considered: P group (three replicate cultures with OVWPE extract diluted in ethanol 20%), E group (three replicate cultures with ethanol 20% alone) C group (three control replicate cultures). 1 ml of VWPE extract (1 mg/ml), 1 ml of ethanol 20% and 1 ml of LB broth were added respectively to the three groups when the OD600 of each culture reached 0.4. Cultures were sampled (2 ml) at the time of treatment (t1), 20 (t2) and 40 (t3) minutes after treatment.
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
Project description:Biotechnology has transformed the production of various chemicals and pharmaceuticals due to its efficient and selective processes, but it is inherently limited by its use of live cells as 'biocatalysts.' Cell-free expression (CFE) systems, which use a protein lysate isolated from whole cells, have the potential to overcome these challenges and broaden the scope of biomanufacturing. Implementation of CFE systems at scale will require determining clear markers of lysate activity and developing supplementation approaches that compensate for potential variability across batches and experimental protocols. Towards this goal, we use metabolomics to relate lysate preparation and performance to metabolic activity. We show that lysate processing affects the metabolite makeup of lysates, and that lysate metabolite levels change over the course of a CFE reaction regardless of whether a target compound is produced. Finally, we use this information to develop ways to standardize lysate activity and to design an improved CFE system.