Composite
dnaKJ sens

Part:BBa_K3773521:Experience

Designed by: Pinar Banu Caglayan   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. To test whether the insert was functional, we needed to check if there was fluorescence production in cells with our circuit. Taking the heat-shocking protocol from Cha et al. (1999) as a model, we conducted a plate reader experiment to measure the fluorescence and OD600 levels of transformed cells that were heat shocked from 37 C to 42 C, cells that were not heat-shocked,untransformed NEB5-alpha cells (negative control), LB supplemented with chloramphenicol (negative control), and NEB5-alpha cells transformed with circuit WM21_013 which we had previously functionally confirmed to be producing green fluorescence constitutively (positive control). Interestingly, the heat shocked cells had fluorescence readings lower than those that were not heat-shocked. This was probably due to the adjustments we made to the model protocol by Cha et al. (1999) where they had originally been cold-shocked at 30 C and then taken to 42 C for the heat-shock for a longer time which may have been why they observed higher fluorescence for the heat-shocked cells in their results. Regardless, our results showed that the heat shocked cells and the non-heat shocked cells had relative fluorescence levels 5- to 10-fold higher than the controls, indicating that the dnaKJ operon promoter is indeed functional and allows for the expression of sfGFP.


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_021_rawfluor.png T--William_and_Mary--Results_WM21_021_nummolecs.png

The following cultures were grown up: one flask of untransformed competent E.coli NEB 5-alpha cells (Untransformed), one flask of dnaJK sensor WM21_021 alone (Sensor Circuit), and two flasks of WM21_021co-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_021 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 the differentially expressed genes informed us that heat-shock response genes dnaK and dnaJ were highly upregulated upon introduction of a heterologous circuit. Our first prediction was that if the sensor circuit was functional, meaning the dnaKJ operon promoter is expressed to produce sfGFP, we expected to observe an increase in average fluorescence in both co-transformed and single-transformed cells when compared to untransformed cells. Our second prediction was that having an additional plasmid, pBbB8k-csg-amylase, would yield higher average fluorescence results in the co-transformed cells (both induced and uninduced) when compared to cells transformed only with the dnaKJ sensor. Lastly, our third prediction was that induction of pBbB8k-csg-amylase would lead to higher average fluorescence values in cells compared to uninduced ones as Birnbaum et al. (2021) had previously characterized the induction-dependent activity of pBbB8k-csg-amylase. With the activated plasmid, we would expect a higher degree of interaction between the host cell and the circuit, which could potentially induce the dnaKJ operon promoter more, indicating that the cells are under stress conditions due to heterologous gene expression. 

Looking at the results of our flask experiments, the average fluorescence is higher at every time point for the induced and uninduced co-transformed, and  single-transformed cell samples compared to the untransformed NEB5-alpha cells. This supports our first prediction that the dnaKJ sensor is indeed functional in cells after transformation and leads to an increased fluorescence production. 

When comparing the average fluorescence values from cells transformed only with the dnaKJ sensor and those co-transformed with the dnaKJ sensor and pBbB8k-csg-amylase (induced and uninduced), the cells with only our sensor circuit had significantly higher average fluorescence at the 6 hour mark compared to the induced pBbB8k-csg-amylase and the sensor circuit containing cells (p-value<0.05). Although a statistical significance was not found at other time points for this comparison, the graph of our fluorescence data shows consistently higher fluorescence for cells with the sensor circuit alone at the 1, 6, 12, and 48 hour marks as well. These results do not support our second prediction that the co-transformed cells would exhibit a higher average fluorescence overall. 

Interestingly, we saw that the co-transformed cells with induced and uninduced pBbB8k-csg-amylase had a higher average fluorescence than the cells with the dnaKJ sensor alone at the 24 hour time point. This led us to consider the growth dynamics of induced and uninduced cells for explaining the possible delay in sfGFP accumulation. Looking at our OD600 data, we observed a higher growth rate for the cells with only the dnaKJ sensor, between hours 1 and 48, compared to both induced and uninduced co-transformed cells for all samples. This could explain the high average fluorescence values for cells with the dnaKJ sensor alone at most time points as those cells are expected to reach the log phase for growth earlier than the co-transformed cells, which could lead to an earlier onset of stress-response at the dnaKJ operon due to unwanted interactions between the host cell and the sensor circuit.

Lastly, evaluating the effect of inducing pBbB8k-csg-amylase on the dnaKJ operon promoter, our results indicated that, only at 24 hours post-induction, the induced cells had a significantly higher average fluorescence compared to uninduced cells (p-value<0.05). At other time points, the average fluorescence of the uninduced cells were consistently higher than those of induced. These results do not support our third prediction that the induction of pBbB8k-csg-amylase would lead to higher expression of the dnaKJ operon promoter than the uninduced samples which would have led to higher fluorescence values.

Overall, these results confirm the functionality of the dnaKJ sensor in both single-transformed and co-transformed cells. Although our results do not support that cells with  plasmids co-transformed induce higher levels of expression at the dnaKJ operon promoter compared to cells with only the sensor circuit transformed, we could explain this difference by inferring temporal variance of the degree of circuit-host cell interactions based on the different growth rates of samples. Additionally, our observation that the uninduced cells led to dnaKJ operon promoter expression at higher levels than the induced cells did not support our prediction of active transcription of a circuit imposing a higher stress, thus higher dnaKJ operon promoter activity. We would have to conduct further experiments with a larger sample size to fully understand the behavior of the dnaKJ sensor in response to co-transformation of two plasmids.

In summary:

  • Our design for the dnaKJ sensor (WM21_021) was confirmed to be functional in responding to an addition of a plasmid which in our design was co-transformation with pBbB8k-csg-amylase into NEB5-alpha cells. 
  • The sensor detected dnaKJ operon promoter expression levels that were higher in cells with only the dnaKJ sensor alone compared to both the induced and uninduced co-transformed cells. 
  • Induced co-transformed cells overall exhibited less expression at the dnaKJ operon promoter compared to the uninduced cells. 

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