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Engineering Escherichia coli to Combat Cancer
Published in Ananda M. Chakrabarty, Arsénio M. Fialho, Microbial Infections and Cancer Therapy, 2019
Carlos Piñero-Lambea, David Ruano-Gallego, Gustavo Bodelón, Beatriz Álvarez, Luis Ángel Fernández
The projected synthetic injector E. coli (SIEC) strain would have integrated in the bacterial chromosome all genes required for the assembly of EPEC injectisomes under the control of an inducible promoter, such as the Ptac, which can be induced with isopropyl-β-d-1-thiogalactopyranoside (IPTG) [99] (Fig. 7.4A). In our approach, gene integration was conducted by a markerless strategy [62] used previously for integration of SAs and lux operon [38]. This strategy renders the resulting engineered bacteria free of antibiotic resistance genes and mobile elements (i.e., plasmids) and allows a stable, controlled expression of the injectisomes.
Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Matthew K. Craft, Grover L. Waldrop
Biotin carboxylase with an N-terminal His tag29, biotin carboxylase carrier protein with an N-terminal His tag30, and carboxyltransferase with a His tag on the N-terminus of the α-subunit31 were expressed separately as previously described, with the following modifications. Cells from glycerol stocks were streaked for isolation on a Luria broth (LB) plate with appropriate antibiotics. A 50 mL culture of LB with an appropriate antibiotic was inoculated with an isolated colony and incubated overnight with shaking at 37 °C. The overnight culture was used to inoculate 0.5 L of LB medium in a 2.0-L flask. The cells were grown at 37 °C, shaking at 200 rpm until mid-log phase. Cells were induced with 50 µM Isopropyl β-d-1-thiogalactopyranoside (IPTG). Shaking was decreased to 150 RPM, and the incubation was continued for 18 h at room temperature. Cells were harvested by centrifugation and resuspended in a minimal amount of Buffer A (5 mM imidazole, 25 mM NaPO4, 500 mM NaCl, pH 8.0). The resuspended cell pellet was then either frozen or purified immediately.
Formation of secondary allo-bile acids by novel enzymes from gut Firmicutes
Published in Gut Microbes, 2022
Jae Won Lee, Elise S. Cowley, Patricia G. Wolf, Heidi L. Doden, Tsuyoshi Murai, Kelly Yovani Olivos Caicedo, Lindsey K. Ly, Furong Sun, Hajime Takei, Hiroshi Nittono, Steven L. Daniel, Isaac Cann, H. Rex Gaskins, Karthik Anantharaman, João M. P. Alves, Jason M. Ridlon
E. coli Top10 [F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG] competent cells from Invitrogen (Carlsbad, CA, USA) were used for manipulation of plasmids, and E. coli BL21(DE3) [F−, ompT, hsdSB(rB− mB−), gal, dcm, rne131 (DE3)] was also purchased from Invitrogen and used for protein expression. 3-oxo-Δ4-LCA, 3-oxo-allo-LCA, 3-oxo-LCA, allo-LCA, LCA, and 3-oxo-DCA were purchased from Steraloids (Newport, RI, USA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Gold Biotechnology (St. Louis, MO, USA). All other reagents were of the highest possible purity and purchased from Fisher Scientific (Pittsburgh, PA, USA).
Efficient drug delivery by novel cell-penetrating peptide derived from Midkine, with two heparin binding sites braced by a length-specific helix
Published in Journal of Drug Targeting, 2022
Yihui Chen, Si Li, Jian Zhao, Xuewei Cao, Fujun Wang
Escherichia coli BL21 (DE3) cells transformed with certain recombinant plasmids were incubated into 30 mL of fresh LB medium containing kanamycin sulphate (50 μg/mL) for 12 h at 37 °C. Then, 4 mL of the primary culture was inoculated into 200 mL of LB culture medium with 50 μg/mL kanamycin. The bacterial cells were cultured at 37 °C until the OD600reached about 0.6–0.8, and then isopropyl‐β‐d‐1‐thiogalactopyranoside (Sangon Biotech, Shanghai, China) was added at a final concentration of 0.5 mmol/L to induce protein expression. Next, bacterial cells were cultured at 16 °C for 16 h. Finally, bacterial cells were collected in centrifuge tubes by centrifugation at 3000×g for 20 min. After suspended in buffer solution (20 mmol/L Tris–HCl, 0.5 mol/L NaCl, 5% glycerol, pH 8.5), Escherichia coli BL21 (DE3) cells were sonicated. Each sample was centrifuged (14,800×g for 15 min at 4 °C) to harvest the supernatant. Each recombinant protein was purified by Ni-NTA affinity chromatography [17]. Protein concentration was determined by a BCA kit. The protein was stored at −80 °C for subsequent experimental research.