Part:BBa_K4174002:Experience

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Applications of BBa_K4174002

William and Mary iGEM 2022

To test the effectiveness of our osmY-sfGFP construct (BBa_K4174002), our team transformed the original MIT iGEM 2006 osmY construct (BBa_J45995), our improved osmY-sfGFP construct, and our improved osmY-mRFP1 construct (BBa_K4174001) in E. coli NEB5-α in a plate reader. The various transformants were grown at 37°C with continuous shaking. For green fluorescence, we used an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The values for green fluorescence are reported below. For information about red fluorescence, see parts page BBa_K4174001.

The data represented in the graph above only includes measurements taken starting from around the 10 hour and 5 minute mark (out of a total growth time of about 19 hours and 35 minutes). In addition, the data shown represents the averages of fluorescence measurements (normalized to OD600) from two experiments.

As seen in the graph above, both the osmY-sfGFP (BBa_K4174002) and MIT iGEM 2006 osmY (BBa_J45995) constructs enter stationary phase right before 14 hours, but our improved osmY-sfGFP construct (BBa_K4174002) is much more fluorescent. The other constructs are our osmY-mRFP1 construct (BBa_K4174001) and untransformed NEB5-α E. coli cells, both of which serve as negative controls for green fluorescence.

Please note that the data in the graph above includes the averages of fluorescence measurements (normalized to OD600) taken from two experiments. For one experiment, we diluted a culture grown overnight in 4 mLs of LB to an OD of 0.1, then loaded the culture into wells to grow overnight. For the other experiment, we inoculated into 1 mL of culture, waited roughly 30 minutes, and loaded the culture into the well plates to grow. For more information on our experimental protocol, please see the Experiments page of the William and Mary iGEM 2022 wiki.

As seen in the image above, qualitative results reveal that our improved constructs are more fluorescent than the original construct (BBa_J45995). Here, a culture of bacterial cells engineered with our osmY-sfGFP construct (BBa_K4174002) is on the far right, and is visibly more green that the culture engineered with the original MIT 2006 iGEM osmY construct (BBa_J45995).

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