Team:Queens Canada/Contribution

Introduction

We compared the amount of GFP expressed under a constitutive promoter (medium promoter, strong RBS) to T7 expression. Some proteins fold better under constitutive promoters; however, nobody had yet directly compared the amount of protein produced between constitutive vs. T7 expression.

See Registry:

Methods

BioBricks were transformed and expressed in E. coli (BL21). BL21 cells were cultured to an OD600=0.6 and 100 uL of culture was transferred into a 96 well plate. Colonies were transferred in quadruplicate. The fluorescence intensity of GFP was measured with a multi-mode microplate reader. The iGEM standardized fluorescence protocol was used for fluorescence measurement standardization.

Figure 1. Fluorescein standard curve used to quantify fluorescent intensity. Slight discrepancy from the linear trend at high concentration is most likely due to oversaturation of signal.

Figure 2. Fluorescein log curve used to quantify fluorescent intensity. This was used to quantify fluorescence of GFP signal.

Figure 3. Fluorescent colonies of E. coli expressing PR4, indicating successful transformation and production of GFP.

Results

We found that the T7 promoter produced about 2.6 times as much fluorescent signal as the constitutive PR4 promoter, indicating that T7 is much more efficient at producing GFP (Fig. 4). Interestingly, the production of GFP under PR4 did not increase beyond the level observed at 4 hours after inoculation. It seems that PR4 leads to an initial production of protein; however, after the initial expression, the promoter seems to be shut off.

Figure 4. Fluorescent intensity of BL21 expressing GFP under T7 (blue), and PR4 (Green). The T7 promoter lead to about 2.6 as much protein being produced over a 16 hour timescale. Fluorescent intensity was determined using the fluorescein standard and logarithmic curves.