Part:BBa_K3773516:Experience

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iGEM21_William_and_Mary

This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.

Our initial confirmation of this part's function was as follows. To test the transcriptional sensor circuits, we transformed them individually into E. coli DH5-alpha. After growing a culture of transformed cells overnight (37°C and 250 RPM), we tested for sfGFP fluorescence using a plate reader by comparing the fluorescence of our circuits to untransformed cells and LB (negative controls) and samples of our translational burden sensor circuit (positive control). The results of our test showed that both circuits are functional and produce high levels of sfGFP. The next step of circuit testing was then to set up an experiment with our translational burden sensor circuits and our test circuit, pBbB8k-csg-amylase, an arabinose-inducible curli fiber production circuit designed by researchers Birnbaum et al. In order to complete this step, we needed to do a cotransformation with the translational burden sensor circuits and our test circuit in E. coli DH5-alpha. After overnight incubation (37°C and 250 RPM), we made subcultures of our overnight cultures and grew all samples in plate reader plates. We compared the fluorescence of samples of our translational burden sensor circuits individually transformed into the host, our translational burden sensor circuits and our test circuit cotransformed into the host (uninduced), our translational burden sensor circuits and our test circuit cotransformed into the host (induced with arabinose), untransformed E. coli DH5-alpha cells, and LB. We measured the fluorescence of all overnight samples, all subcultures right before induction of the test circuit (T0), 1 hour after induction, 6 hours after induction, 12 hours after induction, 24 hours after induction, and 48 hours after induction. We repeated these experiments in flasks a total of three times per translational burden sensor circuit.

As mentioned above, we transformed this circuit into cells alone or alongside pBbB8k-csg-amylase, whose effect on this circuit's expression we hoped to quantify through a change in fluorescence.

The following cultures were grown up: one flask of untransformed competent E.coli DH5-alpha cells (Untransformed), one flask of translational sensor WM21_016 alone (Sensor Circuit), and two flasks of WM21_016 co-transformed with pBbB8k-csg-amylase (arabinose-inducible curli fiber circuit) (Sensor + Test). The sensor circuit and co-transformations were also in E.coli DH5-alpha cells. T = -1 represents measurements taken from these cultures after a growth period of approximately 12 hours, before making subcultures. T = 0 represents measurements taken directly after making subcultures. One flask of WM21_016 co-transformed with pBbB8k-csg-amylase was then induced (Sensor + Test - Induced), while the other remained uninduced (Sensor + Test - Uninduced). T = 1 represents measurements taken 1 hour after the induction step. Measurements were also taken for T=6, T=12, T=24, and T=48 hours post-induction. This process was repeated a total of three times, and the individual recordings are displayed as circles (n=3). The average measurements for each experimental group are displayed as stars and are connected by a line. “Number of molecules” refers to the number of sfGFP molecules per cell, calculated from fluorescence and OD values. P-values for comparison are available on the Results page.

Results:

For the translational burden sensor circuit designed by Ceroni et al. (2015), the uninduced test circuit cotransformed with the sensor circuit produces higher fluorescence than the induced test circuit cotransformed with the sensor circuit at most time points, consistently for all three replicates of the experiment. In addition, for most time points across all three replicates of the experiment, the sensor circuit transformed individually in the host produced higher fluorescence than the cotransformed host (both induced and uninduced). The level of fluorescence produced by our untransformed cells was consistently much lower than that of all transformed cells, remaining at around 5,000 to 8,000 RFU throughout the 48 hour time period. Therefore, we can conclude that this translational burden sensor circuit is functional and that our hypothesis has been supported.

In summary:

• Uninduced test circuit cotransformed with the sensor circuit produces higher fluorescence than the induced test circuit cotransformed with the sensor circuit at most time points
• The level of fluorescence produced by our untransformed cells was consistently much lower than that of all transformed cells
• Uninduced test circuit cotransformed with the sensor circuit produces higher fluorescence than the induced test circuit cotransformed with the sensor circuit at most time points
• We can conclude that this translational burden sensor circuit is functional and that our hypothesis has been supported

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