Difference between revisions of "Part:BBa I746909"

Revision as of 12:13, 19 October 2019

superfolder GFP driven by T7 promoter

This is one of the constructs used to characterise superfolder GFP (see I746916 part description and http://openwetware.org/wiki/IGEM:Cambridge/2008/Improved_GFP for source and other information about this GFP variant)

2019 iGEM team TUDelft

2019 iGEM team TUDelftcharacterized this part in PURE_frex.

Characterization: PR4 vs. T7

Figure 3. 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 4. Fluorescein log curve used to quantify fluorescent intensity. This was used to quantify fluorescence of GFP signal.

Group: Queens_Canada, 2019
Author: Ruben Warkentin

Summary: 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.

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 transfered 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 (https://www.protocols.io/view/calibration-protocol-plate-reader-fluorescence-cal-6zrhf56).

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. 5). 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 5. 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.

Promoter and RBS:
PR4: medium Promoter (J23110) strong RBS (B0034)
T7: T7 Promoter (BBa_I746909)