Team:HZAU-China/Measurement
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Measurement
Overview
Aiming to evaluating the robustness of the two-component-system (TCS)- NarXL, we quantify the its expression flux of various constructs we established. They included adapting different combination of promoter and RBS, two different Escherichia coli strains and inserting an insulator between promoter and RBS. They were tested respectively to identify the activation level of TCS under the identical condition.
We place fluorescence protein at the downstream of the NarXL to act as a reporter gene. Via measuring the fluorescence intensity of bioassay using a microplate reader, the robustness of NarXL can be ultimately quantified. HZAU-China identify the robustness of a bioBrick comprehensively this year, depicting a clear template of how to have a deep insight into the function of a part under different working condition.
Impact of RBS
Firstly, when we placed the same promoter at the upstream of the TCS, aiming to find out the influence that the different RBS exerts towards the nitrate sensing system. The results indicates that, when we have the similar promoter along with different RBS combination, the differences between the strains are quite obvious. The detailed results are listed below.
Promoter: J23109
In the strain E. coli DH5α, the highest expression volume of the outcome products were not affected by the different RBS combination. But when we change our analysis version into the Fold of Change, we found that RBS will significantly affect system’s change. It should be noted that B0033, the weakest RBS in our project, carried out the best performance in the version of fold of change.
Figure 1. The time curve of identical promoter with different RBS combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 2. The Fold of Change of identical promoter with different RBS combination after inducing by nitrate.
In the strain E. coli BL21, different RBS will cause the differences in the climax expression, but there is a similar tendency under the view of Fold of Change.
Figure 3. The time curve of identical promoter with different RBS combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 4. The Fold of Change of identical promoter with different RBS combination after inducing by nitrate.
Promoter: J23110
In the strain E. coli DH5α, the highest expression volume of the outcome products were slightly affected by the different RBS combination. When we change our analysis version into the Fold of Change, we found that RBS will significantly affect system’s change. It should be noted that B0033, the weakest RBS in our project, carried out the best performance in the version of fold of change.
Figure 5. The time curve of identical promoter with different RBS combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 6. The Fold of Change of identical promoter with different RBS combination after inducing by nitrate.
In the strain E. coli BL21, different RBS will cause the differences in the climax expression, but there is a similar tendency under the view of Fold of Change.
Figure 7. The time curve of identical promoter with different RBS combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 8. The Fold of Change of identical promoter with different RBS combination after inducing by nitrate.
Promoter: J23100
In the strain E. coli DH5α, the highest expression volume of the outcome products were seriously affected by the different RBS combination. But when we change our analysis version into the Fold of Change, we found that RBS will have no obvious influence on the system.
Figure 9. The time curve of identical promoter with different RBS combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 10. The Fold of Change of identical promoter with different RBS combination after inducing by nitrate.
In the strain E. coli BL21, different RBS will cause the differences in the climax expression, especially with the weakest RBS, B0033. The fold of Change differs with the change of different RBS.
Figure 11. The time curve of identical promoter with different RBS combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 12. The Fold of Change of identical promoter with different RBS combination after inducing by nitrate.
Effect of promoter
Secondarily, when we placed the same RBS at the upstream of the TCS, aiming to find out the influence that the different promotor exerts towards the nitrate sensing system. The results indicates that, when we have the similar RBS along with different promoter combination, the differences between the strains are quite obvious. The detailed results are listed below.
B0033
In the strain E. coli DH5α, the highest expression volume of the outcome products were affected by the different promotor combination. When we change our analysis version into the Fold of Change, the result becomes more vivid. We found that medium promoter has the best phenotype.
Figure 13. The time curve of identical RBS with different promoter combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 14. The Fold of Change of identical RBS with different promoter combination after inducing by nitrate.
In the strain E. coli BL21, different promoter result in the differences in the climax expression, and there is a similar tendency under the view of Fold of Change.
Figure 15. The time curve of identical RBS with different promoter combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 16. The Fold of Change of identical RBS with different promoter combination after inducing by nitrate.
B0032
In the strain E. coli DH5α, the highest expression volume of the outcome products were seriously affected by the different promotor combination. But when we change our analysis version into the Fold of Change, the result becomes not so obvious.
Figure 17. The time curve of identical RBS with different promoter combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 18. The Fold of Change of identical RBS with different promoter combination after inducing by nitrate.
In the strain E. coli BL21, different promoter result in the differences in the climax expression, and there is a similar tendency under the view of Fold of Change.
Figure 19. The time curve of identical RBS with different promoter combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 20. The Fold of Change of identical RBS with different promoter combination after inducing by nitrate.
B0034
In the strain E. coli DH5α, the highest expression volume of the outcome products were affected by the different promotor combination. But when we change our analysis version into the Fold of Change, the change of promoter J23109 becomes not so obvious.
Figure 21. The time curve of identical RBS with different promoter combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 22. The Fold of Change of identical RBS with different promoter combination after inducing by nitrate.
In the strain E. coli BL21, the highest expression volume of the outcome products were affected by the different promotor combination. But when we change our analysis version into the Fold of Change, the change of promoter J23109 becomes is the most obvious.
Figure 23. The time curve of identical RBS with different promoter combination after inducing by nitrate. Error bars represent the standard error (SE).
Figure 24. The Fold of Change of identical RBS with different promoter combination after inducing by nitrate.
The impact of RiboJ
RiboJ is an insulator placing between the promoter and RBS. We aim to display the restoration situation of the promoter and RBS. Four combinations were tested for each strain.
DH5α
In the strain E. coli DH5α, RiboJ contributes to the restoration of the original combination.
Figure 25. Result of RiboJ insertion of four combinations. A. J23100-B0033; B. J23100-B0034; C. J23109-B0033; D. J23109-B0034
BL21
In the strain E. coli BL21, RiboJ also contributes to the restoration of the original combination, but not so obvious as which in the DH5α.
Figure 26. Result of RiboJ insertion of four combinations. A. J23100-B0033; B. J23100-B0034; C. J23109-B0033; D. J23109-B0034