Part:BBa_K575024

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 PAI1 (3-oxo-C12-HSL), one of the Pseudomonas quorum sensing molecules. The promoter in front of GFP is activated by the combination of PAI1 from the environment and the LasR receptor (produced by this construct). As the graph shows, addition of the autoinducer produces a significant difference in fluorescence compared to the controls. Thus, our construct is successful both in production of LasR and transcription of GFP in the presence of PAI1.

In order to evaluate the suitability of our biosensor constructs for detecting P. aeruginosa, we conducted a series of dose-response studies to characterize our constructs. We also analyzed these data to determine the transfer function and dynamic range of each biosensor system.

Figure 1: Dose response of the PAI-1 biosensor system (LasP+RBS30+GFP and CP+RBS30+lasR). Immediately before the assay, cells were diluted to ensure that they were growing at exponential phase for the experiment. Autoinducer PAI-1 was added at the concentrations indicated, and GFP fluorescence was quantified using an incubated, shaking plate reader. In this plot, fluorescence was normalized to the culture OD to control for cell growth. Samples were run in quadruplicate with standard deviation indicated (Error bars = SD; n=4).

Our first observation was that this construct [lasP+RBS30+GFP, CP+RBS30+lasR] appears to be well-suited for a conducting a binary test to simply determine whether or not P. aeruginosa is present. In every case, the construct follows the same general trend except the negative control (0μM). However, there is is a significant amount of overlapping error bars in Figure 1, so in order to evaluate the statistical significance of this apparent trend, t-tests were conducted as detailed below in Table 1.

Table 1: Statistical analysis of the PAI-1 biosensor response (lasP+RBS30+GFP, CP+RBS30+lasR). 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 (p<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).

In the first 30 minutes, only the sample stimulated with 100μM PAI-1 yielded a response significantly different from the other samples (Table 1A). Our data also indicated that fluorescence per OD changed in each of the samples as time progressed (Table 1B). However, the negative control actually decreased by this measure. This oddity is actually the result of relatively steady total fluorescence and a high rate of cell growth which led to a sharp decrease in fluorescence per OD, as shown in Figure 2 below. In this format, all the samples show similar fluorescence except the 0.1μM, to some extent the 0.5μM, and of course 0μM PAI-1 autoinducer concentrations.

Figure 2: Overall response of the PAI-1 biosensor system (lasP+RBS30+GFP, CP+RBS30+lasR). Here, the data from Figure 1 are presented without normalization, showing total fluorescence (left) and total OD of the cultures.

In order to further characterize this biosensor, we next plotted the steady state fluorescence (per OD) vs. autoinducer concentration to determine the input-output transfer function (Figure 3). As indicated by the analysis and discussion above, in this “binary” biosensor, fluorescence per OD is constant (within about 10% of the mean fluorescence per OD) for all samples we treated with PAI-1 autoinducer, and all samples are significantly distinct from the negative control sample.

Figure 3: Input-output transfer function for PAI-1 biosensor (lasP+RBS30+GFP, CP+RBS30+lasR). Steady-state responses were calculated from the data in Figure 1 and plotted against the input concentration of PAI-1 autoinducer.

LasR/PAI1 Inducible Promoter + RBS (B0030) + GFP, Constitutive Promoter + RBS (B0030) + LasR

Continuous expression of LasR (with RBS B0030), coupled with a LasR/PAI1 (3-oxo-C12-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 58
  Illegal NheI site found at 921
  Illegal NheI site found at 944
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1300
  Illegal AgeI site found at 1497
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 830