Team:Beijing 4ELEVEN/New Part
Our tyrosinase needed a new induced expression system, therefore we constructed our favorite composite part BBa_K3598042 , a part consisting of EilR repressor, EilR PJExD promoters, a T7 terminator, and sfGFP to characterize the system.
The EilR promoter promotes the expression of the EilR, the product of which suppresses the PJExD promoter, thus preventing the expression of the sfGFP. We verified the properties of the cationic dye induced expression system formed by the composite part under the influence of 7 different dyes, by transforming it into E. Coli BL21 and examining the expression of the sfGFP. When cationic dyes are added, the suppression of EilR's expression on the PJExD promoter is eliminated, therefore allowing for the expression of the sfGFP sequence, which makes the cells florescent.
Figure 1. The overall design of our new composite part BBa_K3598042
We tested the efficiencies of different cationic dyes in eliminating the suppression of EilR expression on the PJExD promoter by measuring the florescence of E. Coli BL21 cells after adding different concentrations of the solutions of each dye (Figure 2).
Figure 2. The induction curves of 7 cationic dyes corresponding to different concentrations.
From the result, Crystal violet proves to be most efficient for its subject reaches significant florescence, 36,355 a.u., with concentration of only about 0.1 μM. While Acridine orange proves to be second efficient--although its subject shows rather insignificant florescence in the initial concentration, its effect increases rapidly and reaches 31,376 a.u. at concentration of 5.00 μM, then continues to increase gradually until it exceeds the maximum effect of Crystal violet by a measurable amount. The results of Crystal violet and Acridine orange are separated with the rest for comparison due to their strong efficiency (Figure 3).
Figure 3. The induction curves of Acridine orange and Crystal violet separately.
OMalachite green is less efficient as its subject's florescence increases rather dully with dye concentration, then peaks at 23971 a.u. with concentration of 20 μM. Neutral red's effect experienced a visible increase in concentration intervals 5 and 10 μM, peaks of 22612 a.u. at 50 μM, then begin to decline. To fully examine the influence of Neutral red on the system, a higher concentration is required, therefore it is characterized separately (Figure 4).
Figure 4. The induction curve of Neutral red, a cationic dye which's effect requires higher concentration to examine
Victoria blue R's effects, though not significant, have a constant increasing tendency, which is similar to Methylene blue, with the distinction that the latter increases more slowly. Pyronin Y is the least efficient of all the cationic dyes, as its effects are insignificant and have a low rate of change.
