Team:Hong Kong HKUST/Results
Results
As the stability of our design relies heavily on having a balance in transcription/translation rates and inducer/promoter interactions from different components of the construct, it is necessary to understand how each component behaves in order to fine tune the construct for optimal performance. Therefore, we made smaller constructs to characterize each individual component in terms of promoter strengths, cross-talk between repressors and non-target promoters, and performance under full workload. The protocols used for characterization can be found here.
Characterisation of Component Performance
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. 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 were found to be 4.840e+007 RFU/OD600, 2.275e+007 RFU/OD600, and 4.870e+006 RFU/OD600 respectively, which are all higher than that of 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 the specificity between the repressors and their target promoters. Since all three repressors in our design belong to the same TetR family, it is likely that crosstalks exists; and that should be investigated on. 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 unit upstream to a non-target promoter, in order to drive 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, that is the presence of the repressor transcription unit should be independent of the GFP production cellularly. 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. 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. 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 significant differences 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 is very low. As for crosstalk between TetR and phlfp, we were unable to draw any reliable inference from our results due to a great variation in measurement . Due to time constraint, we did not repeat the characterization.
To investigate crosstalk between tetp and LacI:
To investigate crosstalk between tetp and PhlF:
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. 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. 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, 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 is very low, if any. Interestingly, our results for the comparison between tetp with and without the PhlF transcription unit (Figure 5.) showed that the fluorescence activity driven by tetp with the PhlF transcription unit was higher than that without the PhlF transcription unit. This is unexpected and further investigation is needed to explain this phenomenon.
To investigate crosstalk between lacp and TetR:
To investigate crosstalk between lacp and PhlF:
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. 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. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.
Due to the high error bar generated from our results, we could not make a reliable inference regarding the interaction between TetR and lacp despite the mean fluorescence output of the lacp driven GFP generator being much lower than that of one with a TetR transcription unit upstream to it. Unfortunately, we could not repeat this set of characterization due to time constraint. With regards to PhlF interaction with lacp, our results seem to suggest that PhlF may have a repressing effect on lacp. This needs to be taken into account when fine tuning our design to ensure that the crosstalk doesn't result in instability.
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 be in the full Tristable Switch construct. To visualize 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. This result was obtained by combining 3 characterization data obtained on 3 different days. lacp results were obtained on a separate trial following the same protocol as phlfp and tetp. Error bars represent 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 were found to be 1.071e+007 RFU/OD600, 9.762e+006 RFU/OD600, and 6.543e+006 RFU/OD600 respectively. While phlFp and tetp producing similar levels of fluorescence and lacp exhibiting a significantly lower level of fluorescence. This gives us an insight on how we should fine tune our design.
