high efficiency neb 5 alpha competent e coli cells  (New England Biolabs)

Journal: Nature Communications

Article Title: A self-assembled protein β-helix as a self-contained biofunctional motif

doi: 10.1038/s41467-025-59873-1

Figure Lengend Snippet: a Fluorescence micrographs of HeLa cells transfected with siRNA and pDNA (200 ng) labelled with Alexa 647 and Cy5 (red), respectively, using SaBeH (CR4 for siRNA and CR2 for pDNA). Lipofectamine (LF) is used for comparison. Hoechst 33342 (blue) was used to stain nuclei. b Cell uptake for siRNA (blue) and pDNA (orange) determined by flow cytometry at 3 h post-transfection with SaBeH at CR2 (green) and CR4 (blue) and LF (grey). Data are presented as the (upper) and median (lower) fluorescence with standard deviations for three independent biological replicates ( n = 3): siRNA/mean × 10 4 (CR2: 61.7 ± 6.8; CR4: 97.9 ± 16.9; LF: 33.3 ± 1.7); DNA/mean × 10 4 (CR2: 94.4 ± 3.9; CR4: 75.8 ± 6.3; LF: 6.1 ± 0.4); siRNA/median × 10 4 (CR2: 54.4 ± 7.4; CR4: 87.4 ± 14.5; LF: 13.5 ± 0.3); DNA/median × 10 4 (CR2: 78.2 ± 4.8; CR4: 67.2 ± 5.6; LF: 4.4 ± 0.3). Solid horizontal lines denote median, and upper and lower edges correspond to 75th and 25th percentiles, respectively. According to one-sided ANOVA tests, the counts of transfected HeLa cells for siRNA and pDNA when complexed with LF were significantly lower than those complexed with SaBeH. Statistically significant differences are represented with ** for p < 0.01: mean fluorescence siRNA ( p = 0.0009), mean fluorescence DNA ( p = 6.37 × 10 −7 ), median fluorescence siRNA ( p = 0.00023), median fluorescence DNA ( p = 1.5 × 10 − 6 ). c Schematic of 3D SaBeH scaffold (blue cylinders) and when complexed with pDNA (green cylinders) showing cells without (pink) and with (green) iLOV expression. The Schematic was created using Blender, a free and open-source 3D creation suite. Blender is distributed under the GNU General Public License (GPL), which grants users the freedom to use, modify, and distribute the software for any purpose, including commercially and for education. d Fluorescence micrographs of HeLa cells grown in SaBeH gels without (none) and with the unlabelled, iLOV-encoding pDNA (SaBeH-pDNA). The micrographs highlight cells with significant iLOV fluorescence (green) and are representative of at least 3 independent biological replicates. e Fluorescence micrographs of Escherichia coli and Staphylococcus aureus cells grown over 60 min without (none) and with SaBeH at 100 μM. f Viable cell counts without (grey) and with (black) treatment with SaBeH (100 μM) over 60 min given in percentage. Negative values denote total counts of dead bacterial cells obtained by subtracting counts of viable bacteria grown without SaBeH (100%) from counts of viable bacteria grown with SaBeH. Data are presented as the percentage of the total counts of dead bacteria with standard deviations for three independent biological replicates ( n = 3): SaBeH treated E. coli (−40 ± 19) and S. aureus (−87 ± 43). g Quantification of non-viable E. coli and S. aureus cells treated over 15 and 60 min with SaBeH (white), polymyxin B (light grey) and 70% aq. (v/v) ethanol (dark grey) after subtracting corresponding counts obtained for untreated cells, measured using flow cytometry. Data are presented as the percentages of non-viable bacteria cells in the total number of the cells with standard deviations for three independent biological replicates ( n = 3): SaBeH treated E. coli (15 min: 79.3 ± 2.7; 60 min: 82.7 ± 0.5) and S. aureus (15 min: 93 ± 0.15; 60 min: 94.3 ± 0.03); polymyxin B treated E. coli (15 min: 88 ± 0.49; 60 min: 95.3 ± 0.12) and S. aureus (15 min: 90.5 ± 1.3; 60 min: 94.2 ± 0.07); 70% aq. (v/v) ethanol treated E. coli (15 min: 95.8 ± 0.16; 60 min: 93.9 ± 0.6) and S. aureus (15 min: 94.5 ± 0.74; 60 min: 93.8 ± 1.4). Solid horizontal lines denote median, and upper and lower edges correspond to 75th and 25th percentiles, respectively. Source data are provided as a Source Data file.

Article Snippet: Plasmid transformation was performed in high-efficiency NEB 5-alpha competent Escherichia coli cells (New England Biolabs, Ipswich, UK).

Techniques: Fluorescence, Transfection, Comparison, Staining, Flow Cytometry, Expressing, Software, Bacteria

Journal: Microbial Biotechnology

Article Title: Engineering Genome‐Free Bacterial Cells for Effective SARS ‐ COV ‐2 Neutralisation

doi: 10.1111/1751-7915.70109

Figure Lengend Snippet: SimCell protein–protein Sars‐Cov‐2 RBD competitive ELISA (a) A schematic of the workflow for the RBD‐hACE2 competitive ELISA. Purified nanobody‐displaying SimCells were induced, purified, and diluted to OD600 = 2 with 1× PBS. The diluted SimCells were pre‐incubated with HRP‐conjugated RBD for 1 h statically at 37°C. The pre‐incubation mixture was then added to ACE2‐coated 96‐well microplates. Colorimetric measurement was performed using the chromogenic substrate 3,3′,5,5′‐tetramethylbenzidine (TMB) (Invitrogen), which reacts with HRP on the RBD. Subsequently, the stop solution was added to yield a yellow colour, measurable at 450 nm using a Tecan Spark plate reader. A high OD450nm reading indicates significant binding of HRP‐RBD to ACE2 on the plate, whilst a low or no OD450nm reading suggests minimal or no binding of HRP‐RBD to ACE2. (b) Neutralisation Assays with Wuhan variant RBD: (i) using pNV_Nb6 sfGFP whole‐cell and pNV_Nb6 sfGFP SimCell and (ii) pNV_VE sfGFP whole‐cell and pNV_VE sfGFP SimCell; neutralisation was compared with controls showing no binding and non‐specific counterparts anti‐HER2, labelled as unspecific cells. Wuhan HRP‐RBD concentrations of 0, 4.3, 5.4, 6.5, 8.1, 13, 16.2 and 32.4 nM were used. Both nanobody‐displaying whole cells and purified SimCells were washed and diluted with 1× PBS, then HRP‐RBD pre‐incubated with the washed cells for 1 h at 37°C before addition to the ACE2‐coated plate for 1 h at room temperature. The microplate was washed five times with 1× PBST to remove unbound HRP‐RBD, followed by the sequential addition of an equal volume of TMB and stop solution to yield an OD450nm reading, indicative of RBD‐hACE2 binding. Error bars represent the standard deviation from three biological replicates ( n = 3). (iii) Competitive ELISA with the South African (Beta) variant RBD using pNV_Nb6 sfGFP whole‐cell and bispecific pNV_VE sfGFP whole‐cell; blocking efficiency was compared with controls showing no binding and non‐specific counterparts anti‐HER2, identified as the unspecific cell. South African variant HRP‐RBD concentrations of 0, 4.3, 5.4, 6.5, 8.1, 13, 16.2 and 32.4 nM were selected. Error bars represent the standard deviation from three biological replicates ( n = 3). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: General molecular cloning was performed using NEB 5‐α high‐efficiency competent cells.

Techniques: Competitive ELISA, Purification, Incubation, Binding Assay, Variant Assay, Standard Deviation, Blocking Assay

Journal: Microbial Biotechnology

Article Title: Engineering Genome‐Free Bacterial Cells for Effective SARS ‐ COV ‐2 Neutralisation

doi: 10.1111/1751-7915.70109

Figure Lengend Snippet: Neutralisation curves of anti‐S RBD nanobody displaying mini‐SimCell. (a) Neutralisation curves of NB6 anti‐Spike RBD monomeric nanobody‐expressing ClearColi mini‐SimCells and VE anti‐Spike RBD bivalent nanobody‐expressing ClearColi mini‐SimCells against the Victoria and B.1.351 (Beta) variants. Mini‐SimCells expressing a non‐binding (anti‐HER2) nanobody served as a negative control. Mini‐SimCells were serially diluted 10‐fold dilution for five times, starting from 5 × 10 10 /mL cells down to 5 × 10 5 /mL cells. For the assay, 50 μL of the mini‐SimCell samples were mixed with 200 foci/25 μL/well of viral particles. The XBB‐9 antibody, known for its neutralising capability against both Victoria and Beta variants, was used as a positive control. Error bars represent the average from two plate replicates, each contains two technical replicates ( n = 4).

Article Snippet: General molecular cloning was performed using NEB 5‐α high‐efficiency competent cells.

Techniques: Expressing, Binding Assay, Negative Control, Positive Control