After a period of relentless effort, we have successfully verified the sensing part of our engineered bacteria, marking the completion of the first DBTL cycle experiment.

The result of electrotransformation

Fig.1│After electroporation and recovery, the sample was diluted and spread onto plates containing 100 μg/mL chloramphenicol. The plates were then inverted and placed in an incubator. The appearance of single colonies indicated a preliminary successful transformation of the plasmid into the cells.

Extract plasmids from bacteria and send them to a company for full plasmid sequencing.

Fig.2│Plasmid extraction was performed using the M5 HiPer Multi-color Plasmid Miniprep Kit (with column), and the concentration of the extracted plasmid was measured.

As of before the wiki freeze, the sequencing results were delayed due to internal communication issues at the sequencing company.

After adding the inducer sodium thiosulfate, the fluorescence intensity of the engineered bacteria was measured using a fluorescence microplate reader.

After inducing with different concentrations of thiosulfate and shaking for 12 hours, the OD600 and fluorescence value (Flo) of the bacterial suspension samples were measured respectively to obtain the fluorescence intensity Flo/OD600, which characterizes the activity of the two-component system thsS/R under induction by different concentrations of thiosulfate.

Fig.3│The relationship between the measured fluorescence intensity and the concentration of the inducer sodium thiosulfate is shown in the figure. The F-value of 14.37 is significantly greater than the critical value of 4.95, demonstrating that different concentrations of sodium thiosulfate have a significant impact on fluorescence intensity.

Based on the response results, we can infer that even in the intestinal environment with low concentrations of thiosulfate, the engineered bacteria can still activate and effectively function, while avoiding excessive release of the drug protein. This characteristic enables our engineered bacteria to be extended to the diagnosis and treatment of patients with mild enteritis in practical applications, with higher safety and efficacy.

Furthermore, it can be observed that as the concentration of sodium thiosulfate increases, the fluorescence intensity of the reporter gene gradually decreases. The possible reason for this is that excessively high concentrations of thiosulfate affect bacterial activity, resulting in reduced reporter gene expression. However, at this point, the concentration of thiosulfate has reached above 1mM, which is absolutely beyond the concentration that can be achieved in patients' bodies.