Part:BBa_K3598042

EilR_EilR promoter_PJExD promoter_sfGFP

This part is a tightly repressible induction system, PJExD promoter is regulated by EilR, and several cationic dyes act as efficient low-cost inducers. sfGFP is used for quantitative analysis of the system.

Overall design of the part

To achieve co-expression of adhesive/cohesive protein and tyrosinase, we need a new inducible system different from T7-LacI inducible promoter to control the expression of tyrosinase. Thus, we constructed BBa_K3598042, which consists EilR repressor and PJExD promoter and uses cationic dye as inducer.

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 BBa_K3598042

Experiments and Results of the part

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. Fluorescence of E. coli BL21 with BBa_K3598042 using different dyes as inducer with growing inducer concentration.

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.

Malachite 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 916
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 346
  Illegal AgeI site found at 496
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 954