Team:OUC-China/Demonstrate
1. Introduction
Our project this year focuses on a standardized design principle to be used for modular and tunable riboswitch, which can easily be applied by future teams. We looked at the exsiting riboswitch, where current negative issues like context dependent performance, limited response curve and hard to toggle the on-off state would be addressed as well as solved within our project. The solution to these fundamental but complex issues was introducing Stabilizer, Tuner and asRNA to construct and regulate modular riboswitch, also named RiboLego.
The modular riboswitch we defined consists of the original riboswitch, Stabilizer and Tuner. Stabilizer can protect the structure of riboswitch from damage while Tuner can reduce the expression probability of fusion protein and make improvement of riboswitch function. We test our design principle in different riboswitches including three kinetic switches: Adda riboswitch, Btub riboswitch, cobalamin biosensor, and one thermodynamic switch: FourU riboswitch. What's more, three different kinds of GOI is used including sfGFP, YFP, and mRFP1. The good results show the high universality of our design principles. We believe that we have fulfilled this medal requirement because we can show our system working under real world conditions.
2. Normally express the gene
First, we successfully demonstrated that Stabilizer restored the normal function of riboswitch while Tuner tackled this problem of inclusion body generated by Stabilizer. By fluorescence microscopy, we can clearly observe that Tuner is capable of making GOI express normally.
Figure 1: The fluorescence images represent situation when fluorescence excitation by confocal microscopy. The images show E.coli with 2-AP. Compared with the original Adda riboswitch system and Adda fusion construct, an obvious fluorescence can be observed in modular Adda riboswitch system.
Figure 2: The fluorescence images represent situation when fluorescence excitation by confocal microscopy. The images show E.coli with VB12. Compared with the original Btub riboswitch system and Btub fusion construct, an obvious fluorescence can be observed in modular Btub riboswitch system.
3. Amplify the riboswitch function
Before starting the wet lab work, the core idea of Tuner was successfully modeled by a thermodynamic approach. Using a series of Tuner constructs, we then expand the response curve of modular riboswitch. Five different Tuners were introduced downstream of the activating Adda riboswitch and Stabilizer. Tuners were able to shift the system’s induction response to 2-aminopurine in a manner that correlated with the strength of Tuner.
Figure 3: Histograms show the relative fluorescence expression of sfGFP by microplate reader. Response of each modular Adda riboswitch to 0, 50 and 100 μM 2-aminopurine as compared to the fusion construct(Adda-STA-sfGFP). The five test groups present different fluorescence intensities from high to low, which proves that Tuners have different capabilities. Error bars represent standard deviation of three biological replicates.
We collaborated with four teams which helped us prove the results of Tuner A by experiments in their labs.
Figure 4: The results from other four teams which proved our conclusions. Histograms show the relative fluorescence expression of sfGFP by microplate reader. Response of modular Adda riboswitch including Tuner A to 0, 8, 32 and 250 μM 2-aminopurine. Error bars represent standard deviation of three biological replicates.
To demonstrate the universal applicability of our design principle, the repressing Btub riboswitch was employed that binds adenosylcobalamin. In order to reduce the metabolic burden of cells,we created Tuner H consisting of SsrA degradation tag, which could degrade Stabilizer. Using Tuner A, E and H, we were successfully able to show that we could in fact change the function of riboswitch.
Figure 5: The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. By using three different Tuners, we could change the response curve of Btub riboswitch. Error bars represent standard deviation of four biological replicates.
We also tested our system working by replacing sfGFP with YFP which were introduced downstream of the activating Adda riboswitch, Stabilizer and Tuner A.
Figure 6: The result by microplate reader. The emission of YFP was measured at a wavelength of 527nm when excited at 514nm. Error bars represent standard deviation of three biological replicates. Data was selected when steady state is reached (at least two consecutive subsequent data points do not increase fluorescence).
4. Select the appropriate length of Stabilizer
Guided by math modeling, we determined that the Stablilizer length of Adda and Btub was 150bp. Furthermore, we would prove the effectiveness of our software. So we selected 9bp and 21bp as bad Stabilizers but 81bp and 129bp as good Stabilizers for Adda. The results showed that the length of Stabilizer was changable.
Figure 7: The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. By using four different Stabilizers, we could prove that our software was effective. 9bp and 21bp was too short that can stabilize the structure of Adda riboswitch, leading that the failure of responsive to ligand.
5. Improvement
Using our design principle of modular riboswtch, we were successfully able to improve the cobalamin biosensor created by Paris_Bettencourt team in 2015. They used a riboswitch whose ligand is vitamin B12 to express mRFP1 without its start codon and inserted the first 30bp of the natural gene between them. By confocal microscopy, no fluorescence was be observed because the length of Stabilizer was too short that destroy the structure of riboswitch. By introducing Stabilizer and Tuner A, we constructed an improved cobalamin riboswitch, which can restore his function and express mRFP1 normally.
Figure 8: The results by confocal microscopy, which indicates that our principle can improve cobalamin biosensor successfully. It's obvious that the modular cobalamin riboswitch can express mRFP1.
Figure 9: The fluorescence intensity of mRFP1 by microplate reader during the entire cultivation period. We measured part BBa_K1678007 designed by Paris_Bettencourt in 2015 and the improved circuit designed by us. As shown that, by introducing Tuner A, modular cobalamin biosensor was capable of expressing mRFP1 normally in response to different concentrations of VB12.
6. The thermodynamic switch
Riboswitches can furthermore be classifified into thermodynamic and kinetic switches. We then explored whether our design principles apply to thermodynamic riboswitches. Using Four U, whose temperature threshold is 37℃, we can successfully express sfGFP in 37℃ and 42℃. In this circuit, the first 81bp of mRFP1 was selected as Stabilizer because Four U can control the expression of mRFP1 normally and Tuner A was used. The result shows Tuner can be applied to the thermodynamic riboswitches perfectly!
Figure 10:The results of microplate reader show the working effect of modular Four U element in different temperature.
7. Control the on-off state in real time
By above results, we have demonstrated that Tuners are able to overcome many of the issues preventing widespread use of riboswitches. After constructing modular riboswitches, we have successfully designed antisense RNA to achieve our goals of controling the on-off state in real time. The good results demonstrated our effective approach.
Figure 11: The heat map generated from microplate reader data reflecting the change of fluorescence intensities with and without IPTG. Using our IPTG inducible antisense RNA, we could control the on-off state of Adda and Btub riboswitch.
8. Summary
We believe that we have fulfilled this medal requirement because we have successfully demonstrated that our design principle could expand riboswitch function. Our system could work under realistic conditions. Please see our other pages for more inspiration and results. Additionally, see our medal requirements for information on how we fufilled our medal requirements.
