Part:BBa_K575033

This construct was developed by Northwestern's 2011 iGEM team as part of a Pseudomonas Aeruginosa detector. The device is designed to fluoresce with GFP in the presence of PAI2 (C4-HSL), one of the Pseudomonas quorum sensing molecules. The promoter in front of GFP is activated by the combination of PAI2 from the environment and the RhlR receptor (produced by this construct).

In contrast to the binary detection system, the construct [rhlP+RBS30+GFP, CP+RBS34+rhlR] is well suited for determining the concentration of P. Aeruginosa by detecting and discriminating between different concentrations of autoinducers. Figure 4 below details the fluorescence per OD observed upon the induction of the system with multiple autoinducer concentrations. Unlike the binary detection system, the fluorescence per OD of each of the curves is distinguishable from the other concentrations.

Figure 4: Dose response of the PAI-2 biosensor system 1 (rhlP+RBS30+GFP, CP+RBS34+rhlR). This experiment was conducted as in Figure 1, and strains were exposed to 0 - 100 micromolar PAI-2. Fluorescence and OD were measured every 7.5 min. (Error bars = SD; n=4).

For the most part, overlapping of error bars only occurs when the concentration of the autoinducer increases beyond 15μM which is not relevant for clinical purposes. Notably, autoinducer concentrations between 0μM and 5μM are clear and distinguishable. However, in order to statistically confirm the variance of each concentration curve, t-tests were conducted as detailed below in Table 2.

Table 2: Statistical analysis of the PAI-2 biosensor 1 response (rhlP+RBS30+GFP, CP+RBS34+rhlR). The null hypothesis is that there is no statistical difference between the means of the compared samples. Green cells indicate rejection of the null hypothesis (<0.05), while blue cells indicate failure to reject (p>0.05). (A) Data from each of the initial segments of the curves was compared with the other initial segments (the region before any fluorescence is observed ~30min). (B) Data from the initial segment of the curves (before any fluorescence is observed) was compared with the final steady state fluorescence (last 10 data points). (C) Data from each of the final steady state segments of the curves was compared with the other final steady state segments (last 10 data points).

As we observed with the PAI-1 biosensor, only the 100μM sample significantly induced PAI-2 biosensor 1 within the first 30 minutes (Table 2A).

Table 2b tests the activation of each construct. This is accomplished by comparing the data from the initial segment of the curves (before any fluorescence is observed) with the final steady state fluorescence (last 10 data points). Similar to the observation made in the binary system, each construct changes a statistically significant amount when exposed to the autoinducer. Surprisingly, the 0μM autoinducer sample also has a statistically significant change. However, in this case, the induced constructs produce orders of magnitude more fluorescence than the negative control, so any cell bias introduced by either an OD irregularity is insignificant and can be ignored.

Table 2C compares the data from each of the final steady state segments of the curves with the other final steady state segments (last 10 data points). As can be observed, almost every single steady state fluorescence curve is different than the other. The two exceptions are the 7.5μM-10μM, and the 15μM-20μM autoinducer concentration samples. It is important to note that 10μM-15μM and 20μM-50μM concentration samples have statistically significant responses. In fact, the high degree of discrimination between relative autoinducer concentrations strongly qualifies this construct to be a concentration sensor. The steady state fluorescence per OD is presented in figure 5. The logarithmic regression fits the data quite well, and proves that the construct can potentially be used in as a sensor.

Figure 5: Input-output transfer function for PAI-2 biosensor 1 (rhlP+RBS30+GFP, CP+RBS34+rhlR). Steady-state responses were calculated from the data in Figure 4 and plotted against the input concentration of PAI-2 autoinducer.

RhlR/PAI2 Inducible Promoter + RBS (B0030) + GFP + Constitutive Promoter + RBS (B0034) + RhlR

Continuous expression of RhlR (with RBS B0034), coupled with a RhlR/PAI2 (C4-HSL) inducible promoter, RBS (Part B0030), and a GFP reporter.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 817
  Illegal NheI site found at 840
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1111
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1586
  Illegal BsaI.rc site found at 726