Part:BBa_K4174001:Experience

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Applications of BBa_K4174001

William and Mary iGEM 2022

To test the effectiveness of our osmY-mRFP1 construct (BBa_K4174001), our team transformed the original MIT iGEM 2006 osmY construct (BBa_J45995), our osmY-mRFP1 construct (BBa_K4174001), and our osmY-sfGFP construct (BBa_K4174002) into E. coli NEB5-α cells and grew the various transformants in a plate reader. They were grown at 37°C with continuous shaking. For red fluorescence measurements, we used an excitation value of 584 nm and an emission value of 610 nm. The values for red fluorescence are reported below. For information about green fluorescence measurements, see parts page BBa_K4174002.

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.

Based on the graph above, the bacterial cells engineered with our osmY-mRFP1 construct (BBa_K4174001) appear to have entered stationary phase around 16 hours. As seen in the graph, our osmY-mRFP1 construct produces more red fluorescence than the original osmY construct (BBa_J45995). The other measurements taken are for our osmY-sfGFP construct (BBa_K4174002) and untransformed E. coli NEB5-α cells, both of which serve as negative controls for red fluorescence.

The fluorescence intensity of the untransformed cells appears higher than that of cells transformed with our osmY-mRFP1 construct due to our normalization process. Although the raw fluorescence values of the untransformed cells were consistently lower than the raw fluorescence values of the osmY-mRFP1 circuit, the OD600 values of the untransformed cells were much lower than the OD600 values of the cells transformed with osmY-mRFP1. Therefore, when we normalized by dividing raw fluorescence by OD600, the fluorescence intensity of the untransformed cells appeared to be higher than that of the osmY-mRFP1 transformants.

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 osmY-mRFP1 construct (BBa_K4174001) produces more red fluorescence than the original osmY construct (BBa_J45995). Here, our osmY-mRFP1 construct (BBa_K4174001) is on the far left, and is visibly more red than the original osmY construct (BBa_J45995).

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