Revision as of 10:28, 20 October 2019

Phuc3, one of the series of Phuc

This composite part consists of Pluc BBa_K2913003, conjugation sequence BBa_K2913002, and HucO sequence BBa_K2913001. Phuc3 includes two hucR binding site both in the Pluc and HucO. In the absence of uric acid (or xuanthine), HucR protein binds to Pluc as well as HucO and prevents the binding of RNA polymerase. When uric acid (or xanthine) antagonizes the HucR-Pluc&HucR binding, RNA polymerase initiates a transcriptional process of downstream genes.

Usage and Biology

result

The promoter should be fully activated at the highest uric acid concentration safe to human body (0.465mM). The activity of the promoters were determined by measuring the fluorescence intensity of the enhanced green fluorescent protein (EGFP). The results showed that the Phuc2 had the highest sensitive and activity at the conditions of 0.1mM and 0.01 mM of uric acid. Then, the EGFP coding sequence was replaced by the uricase gene to check whether the module could sense and metabolize the uric acid. As shown in Fig.5, the control group kept constant uric acid levels, but the experimental group displayed reduced uric acid concentrations in a time-dependent manner in the first 3 h of incubation. After that, the uric acid levels kept constant. The data suggested that the expression of uricase was induced at high concentrations of uric acid and repressed at its relatively low concentrations. The result indicated that the expression of uricase was under the control of Phuc2 responsive to the concentration of uric acid.

Fig. 3 Comparison of induction strength of the promoter Phuc1 (circle), Phuc2 (square), and Phuc3 (triangle) by measuring green fluorescence of EGFP at 0.1 mM of the inducer uric acid (with 1:100 dilution of culture inoculation). Error bars represent standard deviations.

Fig. 4 Comparison of induction strength of the promoter Phuc1 (circle), Phuc2 (square), and Phuc3 (triangle) by measuring fluorescence of EGFP at 0.01 mM of the inducer uric acid (with 1:100 dilution of culture inoculation). Error bars represent standard deviations.

Fig.5 The cultured bacteria were transferred into M9 medium containing 8 mM uric acid. Samples were collected every 30 min to measure the concentrations of uric acid. The control group was the Nissle bacteria while the experimental group was the Nissle bacteria with the uric acid regulation module. Error bars represent standard deviations.

Sequence and Features