Characterization of Small Constructs

Relative Strengths of Promoters

To compare the relative strengths of the three promoters, phlFp, tetp, and lacp, we ligated each promoter to a GFP generator, BBa_E0240, and measured the relative fluorescence unit (RFU) produced by each construct. The following constructs were built:

Figure 1. Comparison on the strength of phlFp, tetp and lacp. Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

The activities of phlFp, tetp, and lacp were measured and compared in RFU and normalised with OD₆₀₀. The activities of phlFp, tetp, and lacp was found to be 4.840e+007 RFU/OD₆₀₀, 2.275e+007 RFU/OD₆₀₀, and 4.870e+006 RFU/OD₆₀₀ respectively higher than the negative control, with phlFp exhibiting the strongest promoter strength, followed by tetp, and lacp.

Crosstalk between Repressors and Non-target Promoters

The fidelity of our design relies on specificity between the repressors and their target promoters. Especially since all three repressors in our design belong to the same TetR family, crosstalk between the repressors and non-target promoters should be investigated. We ligated the constitutive promoter, BBa_J23101 to transcription units of each repressor to achieve a constitutive expression of the repressor protein. We then inserted the resulting composite upstream to a non-target promoter driving the expression of the GFP generator, BBa_E0240. The following constructs were built:

To investigate crosstalk between phlFp and LacI:

To investigate crosstalk between phlFp and TetR:

Ideally, there should be no crosstalk between the repressors and non-target promoters which means that the presence of the repressor transcription unit should have no effect on the cell's GFP production. In that scenario, the GFP production of the two constructs above would be similar to that of a GFP generator driven by phlFp. Therefore, we compared the fluorescence activity of the two constructs above against a GFP generator driven by phlFp (the construct that was used to characterize phlFp promoter strength).

Figure 2. Comparison between the fluorescence expression levels of phlFp with and without LacI transcription unit. Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

Figure 3. Comparison between the fluorescence expression levels of phlFp with and without TetR transcription unit Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

Our results showed that there was no signifcant difference between the fluorescence activity of phlFp with and without the LacI transcription unit. Therefore, it is likely that the crosstalk between LacI and phlfp in our system would be very low. As for crosstalk between TetR and phlfp, we were unable to draw any reliable inference from our results due to the high error bar. Due to time constraint, we did not repeat the characterization.

To investigate crosstalk between tetp and LacI:

We compared the fluorescence activity of the two constructs above against a GFP generator driven by tetp (the construct that was used to characterize tetp promoter strength).

Figure 4. Comparison between the fluorescence expression levels of tetp with and without LacI transcription unit. Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

Figure 5. Comparison between the fluorescence expression levels of tetp with and without PhlF transcription unit Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

From our results, it appears that there was very little difference between the fluorescence activity of tetp with and without the LacI transcription unit. Therefore, it is likely that the crosstalk between LacI and tetp in our system would be very low, if any. Interestingly, our results for the comparison between tetp with and without the PhlF transcription unit (Figure 5.) showed that the fluroescence activity driven by tetp with the PhlF transcription unit was higher than that without. This is unexpected and further investigation is needed to explain this phenomenon.

To investigate crosstalk between lacp and TetR:

We compared the fluorescence activity of the two constructs above against a GFP generator driven by lacp (the construct that was used to characterize lacp promoter strength).

Figure 6. Comparison between the fluorescence expression levels of lacp with and without TetR transcription unit. Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

Figure 7. Comparison between the fluorescence expression levels of lacp with and without PhlF transcription unit Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

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Performance of Promoters under Full Workload

We were also interested in studying how well the promoters perform when they are driving the expression of two repressors on top of the reporter gene just like how they would in the full Tristable switch construct. To get an idea of how well the promoters would perform under full workload, the following constructs were built:

To investigate performance of phlFp:

To investigate performance of tetp:

To investigate performance of lacp:

Figure 8. Performance of phlFp, tetp and lacp Under Full Workload. Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. lacp results were obtained on a separate trial but followed the same protocol as phlfp and tetp. Error bar present SD from 3 biological replicates.

The fluorescence activities of phlFp, tetp, and lacp under full workload were measured and compared in RFU and normalised with OD₆₀₀. The activities of phlFp, tetp, and lacp was found to be 1.071e+007 RFU/OD₆₀₀, 9.762e+006 RFU/OD₆₀₀, and 6.543e+006 RFU/OD₆₀₀ respectively, with phlFp and tetp producing similar levels of fluorescence and lacp exhibiting a significantly lower level of fluorescence.