Composite
clpB senso

Part:BBa_K3773517:Experience

Designed by: Justin Berg   Group: iGEM21_William_and_Mary   (2021-10-15)


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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 (WM21_013, which we had previously seen to fluoresce). Cells from both colonies containing WM21_017 fluoresced about half as much as the positive control, which was not as high as we had hoped. Additionally, we heat shocked an additional sample of cells from each colony to confirm the circuit’s sensitivity to some form of stress, as authors who have used the promoter in a circuit before have seen an increase in fluorescence after heat shock (Cha et al., 1999). We placed the cells, which were initially at 37ºC, into a 42ºC heating block for 20 minutes. We then measured green fluorescence in the plate reader and saw that, on average, Colony 1 induced cells fluoresced about 10% more than uninduced cells. Colony 2 induced cells fluoresced about 60% more. It is worth noting that if we had waited for a longer time after heat shock before measuring fluorescence, the increase would likely have been higher (Cha et al., 1999). Additionally, when we cotransformed WM21_017 with pBbB8k-csg-amylase into NEB5-alpha cells, we saw that fluorescence was consistently higher than when WM21_017 was transformed alone into cells. We had designed this circuit to show increased fluorescence when cotransformed with a second circuit such as pBbB8k-csg-amylase.


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.

T--William_and_Mary--FigLegendRegistry.png T--William_and_Mary--Results_WM21_017_rawfluor.png T--William_and_Mary--Results_WM21_017_nummolecs.png

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_017alone (Sensor Circuit), and two flasks of WM21_017 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_017 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 clpB was frequently upregulated upon introduction of a heterologous circuit. As a result, our first prediction was that when transformed into cells alongside another circuit such as pBbB8k-csg-amylase, the clpB sensor circuit would fluoresce more than when it was transformed into cells alone. Additionally, our second prediction was that the clpB sensor would 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. 

Our cultures in fact appeared to fluoresce more on average when only the clpB sensor was present than when it was alongside pBbB8k-csg-amylase, refuting our first prediction. On average, all of our cultures that were transformed with the sensor circuit alone did increase in fluorescence over time, confirming the first half of our second prediction; however, our cotransformed cultures did not increase faster than those containing the clpB sensor alone, indicating that the circuit was not fluorescing more due to to the presence of pBbB8k-csg-amylase. Finally, of our cotransformed cultures, induced cells were more often higher in fluorescence than uninduced cells. This somewhat confirms our third prediction, although the difference was small, inconsistent across replicates, and not evident after converting raw fluorescence values to number of sfGFP molecules per cell. 

It is important to note that these results are not statistically significant (p-value>0.05), however, largely because we were only able to obtain three replicates of data. We performed a t-test to evaluate the difference 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 sensor circuit. Only B was consistently statistically significant (p-value<0.05) across time points. Although not 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 average fluorescence of cells containing the sensor circuit alone, we conclude that the circuit does not successfully report an increase in PclpB expression when a second circuit is present. Additionally, it appears that pBbB8k-csg-amylase is so burdensome to the expression of the clpB sensor that rather than causing its promoter to be upregulated as intended, it uses up cellular resources needed to produce sfGFP. One explanation is that this version of the clpB sensor simply does not produce enough fluorescence to compete with pBbB8k-csg-amylase. We notice that WM21_018, which appeared four times as fluorescent as WM21_017 during functional confirmation, does show greater fluorescence when cotransformed alongside pBbB8k-csg-amylase than otherwise. It may be that circuits that produce a low level of sfGFP are more easily outcompeted by pBbB8k-csg-amylase. 

In summary:

  • Unexpectedly, cotransformations fluoresced less than sensor-alone cells
  • Transformed cells expectedly increased in fluorescence over time
  • Induced cotransformations may have fluoresced more than uninduced cotransformations, which would be expected
  • We conclude that this circuit is not a successful reporter of differential expression of clpB; it is likely burdened by and unable to compete with pBbB8k-csg-amylase for cell resources

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