Team:Gaston Day School/Composite Part

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Composite Parts

Descriptions														Parts
Cadmium detector linked to RFP														BBa_K2994100
Temperature Sensitive GFP Production Construct														BBa_K2994001

Cadmium Detector

•Designed to be used with an Arduino RGB color detector or to be used in situations where a visible color change is needed

•RFP was chosen as the indicator because the red color is visible without the use of specialized equipment

Figure 1. Cadmium Detector BBa_K2994100. K174015 is a cadmium sensor composed of a cadmium sensitive promoter and a cadmium binding protein; B0035 is a ribosomal binding site; E1010 is the RFP; and B0014 is a terminator.

*Testing the Cadmium Detector:

Cultures were grown for 24 hours in the presence of 0, 0.25, or 0.75 mM cadmium. Absorbance was measured using a Vernier SpectroVis Plus and data was collected and analyzed using Microsoft Excel’s t-test function. Results are reported as absorbance at 517 nm normalized using absorbance at 700 nm. Results for both 0.25 and 0.75 mM cadmium are significantly different from the negative control at the p<0.01 level (n=11).

Figure 2. Absorbance at 517nm per OD700. Results are significantly different from the control (p<0.01).

Temperature Sensitive GFP Production Construct

•Efficient Temperature Sensitive GFP Production Design

•Initial observation shows that gTEMP changes its intensity when temperature changes. Further quantitative testings are required to confirm these observations.

Figure 3. Temperature Sensitive GFP Production Construct. J23100 is a strong constitutive promoter; B0035 is a ribosomal binding site, and K2994000 is the coding region of the temperature sensitive GFP.

Figure 4. Gel for gTEMP. Lane 1 is the long ladder and Lanes 2-8 are K2994001. The white box shows the area where the bands are located in. The bands are in the correct size.

Figure 5.Test Data for gTEMP. Data were collected using a SpectroVis Plus from Vernier with the excitation wavelength set at 405nm and the peak emission from 410-415nm. If the construct were working, we would expect a linear increase in absorbance as temperature increases. These points are clearly not linear and not significantly different from each other (n=8). Bars represent average ± SEM.