Part:BBa_K3773523: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. We sent our inserts for sequencing and used the Benchling tools to confirm their successful alignment with our ordered sequence. Additionally, we confirmed that the circuit produced green fluorescence by inoculating cells from our two glycerol stocks overnight and placing them in the plate reader along with two negative controls (LB alone and untransformed NEB5-alpha cells) and a positive control (BBa_K3773513, which we had previously seen to fluoresce). Each glycerol stock came from a different initial colony of cells containing this circuit. Although the positive control failed to fluoresce in this case for unknown reasons, cells from both glycerol stocks containing BBa_K3773523 fluoresced far more than the negative controls. Additionally, the positive control did fluoresce during functional confirmation of the clpB and hslVU sensors; when compared to these fluorescence values, lon produced fluorescence that was about half as high as that of the positive control for both colonies.

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 NEB 5-alpha cells (Untransformed), one flask of protease lon sensor WM21_023 alone (Sensor Circuit), and two flasks of WM21_023 co-transformed with pBbB8k-csg-amylase (arabinose-inducible curli fiber circuit) (Sensor + Test). The sensor circuit and co-transformations were also in E.coli NEB 5-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_023 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:

Our analysis of differentially expressed genes told us that lon was frequently upregulated upon introduction of a heterologous circuit. As a result, we expected that when transformed into cells alongside another circuit such as pBbB8k-csg-amylase, WM21_023 would fluoresce more than when it was transformed into cells alone. Second, we expected the lon sensor to produce increasing fluorescence over time, especially in cotransformed cells, as we believed pBbB8k-csg-amylase would become more productive and thereby cause more effects on the cell over time. Third, we predicted that cotransformed cells would fluoresce more when induced than otherwise. This is because we expected pBbB8k-csg-amylase, which is inducible by arabinose, to have more effects on regular cell functions if its expression levels were increased. 

On average, our cultures in fact appeared to fluoresce far more, and increase in fluorescence far faster, when only the lon sensor was present than when it was alongside pBbB8k-csg-amylase. This contradicts our first prediction. While average fluorescence appeared to increase over time for all transformed cells, supporting our second prediction, when converted to number of sfGFP molecules per cell, this was not the case; average sfGFP increased for the sensor circuit alone and from about 6 to 12 hours after induction for induced cells, but average fluorescence decreased until 48 hours for uninduced cells. Finally, of our cotransformed cultures, induced cells were on average consistently higher in both fluorescence and number of sfGFP molecules per cell than uninduced cells after 6 hours. This confirms our third prediction.

It is important to note that these results are not totally statistically significant, however, largely because we were only able to obtain three replicates of data. We performed a t-test to evaluate the difference in fluorescence between: A) uninduced versus induced, cotransformed cells; B) induced cells versus untransformed cells, the negative control; and C) induced, cotransformed cells versus cells containing only the lon sensor. Only B and C were consistently statistically significant (p-value<0.05), and only after one or six hours, respectively, when fluorescence values diverged most strongly. Although not fully statistically significant, we were able to observe the aforementioned, suggested trends, which might become clearer with more testing.

Because the average fluorescence of cotransformed cells both was lower than and increased slower than the fluorescence of cells containing the lon sensor alone, we conclude that the circuit does not successfully report an increase in Plon expression when a second circuit is present. pBbB8k-csg-amylase may be so burdensome to the expression of the sensor circuit that rather than causing its promoter to be upregulated as intended, it uses up cellular resources needed to produce sfGFP. The lon sensor circuit showed only about half as much fluorescence as WM21_018 during functional confirmation, so it may be that the lon sensor simply does not produce enough fluorescence to compete with pBbB8k-csg-amylase. However, as time passed, the fluorescence for the lon sensor alone here reached even higher values (above two million RFU) than those obtained for WM21_018 alone. Therefore, there may be some other factor at play that prevents the lon sensor circuit from maintaining access to cell resources. However, induced cells are clearly more fluorescent than uninduced cells, suggesting that this circuit may be able to report increased lon expression caused only by induction of a circuit.

In summary:

• The cotransformations unexpectedly fluoresced less than sensor-alone cells.
• Transformed cells expectedly increased in fluorescence over time
• Induced cotransformations expectedly fluoresced more than uninduced cotransformations.

We conclude that this circuit is a successful reporter of differential expression of clpB when comparing induced to uninduced cells, but not when comparing induced cells to those containing only the sensor.

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