Part:BBa_K3773524: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 (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 colonies containing the hslVU sensor fluoresced at a similar level to the positive control. 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 (Lien et al., 2009). 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 30% more than uninduced cells. Colony 2 induced cells fluoresced about 20% more. As a result, we decided to use our Colony 1 as our main source of cells; additionally, its MiniPrep had the highest concentration of the two MiniPrepped colonies. 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 (Lien et al., 2004).

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 hslVU 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 DE gene analysis revealed that hslVU would be upregulated in response to the presence of an orthogonal circuit within the cell. We predicted that the pBbB8k-csg-amylase circuit would decrease the orthogonality of the cell, and the cotransformation of both the pBbB8k-csg-amylase circuit plasmid and our sensor circuit plasmid into a cell would have lower orthogonality than the insertion of the sensor circuit by itself, leading to higher average fluorescence values. Our second prediction was that we would observe higher average fluorescence in the transformed and cotransformed cells, as compared to the untransformed cells that did not contain the sfGFP coding region. Our final prediction was that average fluorescence values in the transformed and cotransformed cells would increase over time, as the pBbB8k-csg-amylase circuit imposed greater and greater strain on the host circuit functions.

For our statistical analysis, we performed three sets of unpaired t-tests at each time point we measured. These three comparisons were: induced vs. uninduced, induced vs. untransformed, and induced vs. the sensor alone. We were unable to demonstrate statistical significance between the uninduced and induced cells, indicating that induction does not make much of a difference in the orthogonality of this circuit, as measured by this sensor. The induced cotransformed cells had statistically significant average fluorescence values from the untransformed cells at certain time points (p-value<0.05). However, whether the uninduced cells fluoresced more than the cells containing only the sensor circuit varied from replicate to replicate. We were also unable to find statistical significance between the induced contransformed cells and the cells containing only our sensor circuit.

It was difficult to achieve statistical significance in our data due to a low number of trials, however we observed several interesting trends. The untransformed NEB5-alpha cells stayed at a consistently low fluorescence given their lack of a fluorescence gene, establishing them as a good negative control for our experiment. We also generally observed an increase in fluorescence at a decreasing rate over time after subculturing at t0.

We likely obtained these results because the marginal strain imposed on the host cell by the pBbB8k-csg-amylase circuit was not higher than the strain already imposed by our sensor circuit. The high average fluorescence values in transformed and cotransformed cells indicate that our sensor circuit was functional, but we would have to conduct additional trials to determine whether or not our sensor circuit was truly able to sense the difference in orthogonality due to the circuit of interest. Further trials would also elucidate whether the temporal differences in significance persist when comparing induced vs. untransformed cells.

In summary:

• The hslVU sensor circuit cotransformed with the induced pBbB8k circuit was expected to have increased fluorescence over time, with greater fluorescence than the culture of hslVU with uninduced pBbB8k and the culture of hslVU alone.
• The average fluorescence of the induced cotransformed cells was significantly higher than that of the untransformed negative control.
• Average fluorescence for all cells containing the hslVU sensor circuit generally increased over time.
• We were unable to statistically determine whether there was a difference in average fluorescence between the transformed and cotransformed cells, regardless of the state of induction.
• These results suggest that the pBbB8k-csg-amylase circuit may not have a more detrimental effect on the cell than that imposed by the hslVU sensor circuit by itself.

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