Journal: bioRxiv

Article Title: Engineering signal peptide-mediated recombinant protein secretion in Komagataeibacter rhaeticus for biological Engineered Living Material applications

doi: 10.64898/2025.12.15.694344

Figure Lengend Snippet: An overview of SP library construction and SP characterization. (A) General modular construction of secretion strains, cloning the N-terminal SP library fused to β-lactamase in an expression cassette transformed into both E. coli and K. rhaeticus and characterized using a 96-well nitrocefin assay. (B) The rate of reaction of β-lactamase from E. coli supernatants, comparing the strains constructed with the various SPs fused to β-lactamase; two control strains were included: β-lactamase with no SP (Bla NO SP) and a GFP expression control (GFP); assay activity of all strains were normalized to the negative control. Rate of nitrocefin hydrolysis, expressed as the change in signal over time (D[S]/Dt −1 ), with units of signal increase per second (t= - ).

Article Snippet: Nitrocefin (chromogenic β-lactamase substrate) (Merck) was prepared as a 10 mg/mL stock solution in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions.

Techniques: Cloning, Expressing, Transformation Assay, Construct, Control, Activity Assay, Negative Control

Journal: bioRxiv

Article Title: Engineering signal peptide-mediated recombinant protein secretion in Komagataeibacter rhaeticus for biological Engineered Living Material applications

doi: 10.64898/2025.12.15.694344

Figure Lengend Snippet: Analysing SP efficiency in K. rhaeticus using medium and high strength promoters. Endpoint nitrocefin assay conducted on K. rhaeticus strains after 72 hours. ( A ) The data shown is the endpoint 480 nm absorbance measurement taken after the nitrocefin incubation for 48 hours. The WT (wild type) and Bla No SP (cytoplasmic β-lactamase) strain are negative controls. The engineered strains are the various N-terminal SPs fused to β-lactamase (n=3), under medium strength promoter. (B) The rate of reaction of the high strength variants of YwmC and EPR, showing the activity in both cell pellet and supernatant, cytoplasmic β-lactamase control. Absorbance measured at 480 nm. Data points represent the mean of triplicate samples ±1 SD. One-way ANOVA, P=<0.0001, showing significance of data as P <0.05. Rate of nitrocefin hydrolysis, expressed as the change in signal over time (D[S]/Dt −1 ).

Article Snippet: Nitrocefin (chromogenic β-lactamase substrate) (Merck) was prepared as a 10 mg/mL stock solution in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions.

Techniques: Incubation, Activity Assay, Control

Journal: bioRxiv

Article Title: Engineering signal peptide-mediated recombinant protein secretion in Komagataeibacter rhaeticus for biological Engineered Living Material applications

doi: 10.64898/2025.12.15.694344

Figure Lengend Snippet: (A) Images of BC pellicles grown from engineered K. rhaeticus β-lactamase secretion strains, assaying 4 SPs (YwmC, EPR, SP5, SP4) and a control with no SP (Bla). Conversion of nitrocefin into its hydrolyzed red form over 120 minutes is visual evidence of enzyme activity. After growth, pellicles were washed so that they only retain enzymes within BC. (B) Nitrocefin hydrolysis assay showing the effect of the treatment of OSB on functional activity within BC spheroids. Strains tested were the no-secretion control (Bla) and the strain with YwmC SP. Spheroids were either untreated (UT) or OSB treated (OSB) over the period of one hour. ( C) The rates of nitrocefin hydrolysis were then calculated from the assay shown in (B) , revealing the difference between functionalized and non-functionalized spheroids. Data shown is an average of triplicate samples with ± 1 SD. Rate of nitrocefin hydrolysis, expressed as the change in signal over time (D[S]/DT-1), with units of signal increase per second T= minutes.

Article Snippet: Nitrocefin (chromogenic β-lactamase substrate) (Merck) was prepared as a 10 mg/mL stock solution in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions.

Techniques: Control, Activity Assay, Hydrolysis Assay, Functional Assay

Journal: Molecules

Article Title: Kaempferol and Kaempferin Alleviate MRSA Virulence by Suppressing β-Lactamase and Inflammation

doi: 10.3390/molecules30204132

Figure Lengend Snippet: Kol and kin inhibit the activity of S. aureus β-lactamase by binding to the active center. ( a ) The structure of kol and kin. ( b ) The activity of β-lactamase when treated with or without tested compounds. β-lactamase protein was co-incubated with various concentrations of kol or kin, then nitrocefin was added, and samples were co-incubated. Inhibition was determined by measuring absorbance at 492 nm. Data are present as means with SD, n = 3, ** represents p ≤ 0.01. ( c ) The binding mode, the affinity, and the potential binding sites between β-lactamase and kol or kin ( d ). Protein was colored by chain (cyan), kol (colored with green) and kin (colored with blue) are shown as sticks.

Article Snippet: Nitrocefin, which is the substrate of β-lactamase, was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

Techniques: Activity Assay, Binding Assay, Incubation, Inhibition