Part:BBa_K3773518: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, after inserting te circuit into the 1C3 plasmid backbone and successfully transforming into NEB 5-alpha cells, we confirmed that the circuit produced green fluorescence. We did so 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 NEB 5-alpha cells) and a positive control (WM21_013, which we had previously seen to fluoresce). Each glycerol stock had been derived from a different colony of cells containing this part. Cells from both glycerol stocks containing BBa_K3773518 fluoresced about twice as much as the positive control.

When we cotransformed BBa_K3773518 with pBbB8k-csg-amylase into NEB 5-alpha cells, we saw that fluorescence was sucessfully higher over time for cotransformed cells than cells with BBa_K3773518 alone. This suggests that, unlike BBa_K3773517, BBa_K3773518 successfully responds to the presence of pBbB8k-csg-amylase with increased sfGFP expression.

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. Pleas note that WM21_018 is another name for this part, BBa_K3773518.

The following cultures were grown up: one flask of untransformed competent E.coli NEB 5-alpha cells (Untransformed), one flask of heat shock sensor WM21_018 alone (Sensor Circuit), and two flasks of WM21_018 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_018 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 of our wiki.

Results:

We had the same expectations for WM21_018 as we had had for the earlier version of it, WM21_017. First, we expected this new version of the clpB sensor to fluoresce more in cotransformed cells than cells with the sensor alone. Second, we predicted that fluorescence of all transformed cells would increase over time, and that the fluorescence of cotransformed cells would increase faster than that of those with the sensor alone. Finally, we expected cotransformed cells to fluoresce most after being induced. 

Uninduced, cotransformed cells did generally appear to fluoresce more on average than cells containing the sensor circuit alone, supporting the first prediction. Induced, cotransformed cells appeared to have a similar result only after converting fluorescence to number of sfGFP molecules per cell. This is likely because induced cells took longer to grow in Replicates 1 and 2, according to our OD600 measurements at each time point. It may be that with more replicates, this issue would be resolved, but it is likely that the burden imposed on an induced, cotransformed cell places limitations on growth rate. The low average fluorescence of induced cells contradicts our third prediction. Average fluorescence of all transformed cells did increase over time, fulfilling our second prediction, although fluorescence of induced cells increased slower than that of other cells, likely due to the previously mentioned reasons. Fluorescence later decreased on average for uninduced cells and cells with the sensor circuit alone; these cells were likely unable to sustain a high production of sfGFP over such a long period of time, especially with the presence of two circuits. 

As with the earlier version of this circuit, it is important to note that many of the differences between fluorescence measurements were not statistically significant (p-value>0.05) according to a t-test. Once again, only the comparison between induced versus untransformed cells returned a p-value of less than 0.05 for multiple time points. If we were able to perform more replicates, the aforementioned trends suggested by the data might become more clear. 

Because cotransformed cells produced more sfGFP over time than WM21_018 alone, we concluded that WM21_018 is an improved version of WM21_017, and could plausibly be used to report the unintended interactions occurring between a secondary circuit and the host cell. This success, while limited by our number of replicates, is likely due to the high fluorescence of WM21_018, which was shown to fluoresce about four times as much as WM21_017 during functional confirmation. This allows us to measure the upregulation of the clpB promoter when a circuit such as pBbB8k-csg-amylase is present.

In summary:

• Only uninduced cotransformations clearly fluoresced more than sensor-only cells
• Transformed cells expectedly increased in fluorescence over time
• Induced cotransformations unexpectedly fluoresced less than uninduced cotransformations
• We conclude that this circuit is a successful reporter of differential expression of clpB; although induced cells grow slower and therefore fluoresce less, this is an improvement upon WM21_017.

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