Part:BBa_K2904000
Tuner A
Design
Background of 2019 OUC-China's project——RiboLego
Due to context-dependent performance and limited dynamic range, the widespread application of riboswitches is currently restricted. By replacing its original ORF with a new one, the structure of an aptamer domain can be subtly disrupted, resulting in a loss of ligand response. So riboswitch is still not be considered as a “plug and play” device. To tackle these problems, our project focuses on a standardized design principle to be used for modular and tunable riboswitch. 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.
The construction of this part
We defined a Tuner element to include a repressing region, a RBS region and a coupled junction region. The repressing region is the reverse complement of a subsequence of the RBS region so that Tuner can form a hairpin with appropriate
∆G. The stop and start codon fused in the junction region. Ribosomes recruited by the upstream riboswitch can open up the hairpin of Tuner before dissociation at the stop codon in the junction region. Additional ribosomes can then assemble at the Tuner RBS and initiate translation at the first start codon of the introduced gene of interest. Therefore, Tuner can facilitate tuning of a riboswitch’s response and help GOI express normally.
The following diagram shows the structure of Tuner A and we marked each region clearly.
Result
Overview
Using this part, we could engineer many modular riboswitches. In order to validate the effect of Tuner A, we utilized four kinds of riboswitches, including Adda riboswitch, Btub riboswitch, cobalamin riboswitch and FourU. The results demonstrate that Tuner A can help achieve tunable and efficient gene regulation. Besides, we also used Tuner A to explore the source and length of Stabilizer. [http://2019.igem.org/Team:OUC-China/Results More results are on this page!]
Modular Adda riboswitch
First, we employed Adda riboswitch, which can regulate the expression of adenosine deaminase by binding 2-aminopurine in Vibrio vulnificus.The first 150bp of adenosine deaminase was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. We used Tuner A to construct modular Adda riboswitch and sfGFPas the reporter gene to reflect output of our system.
For the sake of functional test, other 2 circuits are set, Adda-sfGFP and Adda-Stabilizer-sfGFP, which also were under control of the tetracycline promoter. By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the adenine riboswitch had sfGFP introduced directly. The direct fusion of sfGFP to Stabilizer yielded very clear inclusion bodies, manifested as distinct spots present at one pole of the cell which are formed by misfolded insoluble proteins. By comparison, the modular Adda riboswitch yielded soluble working protein since Tuner A has the ability to insulation the target gene from Stabilizer.
The qualitative experiment is not enough to analyze the modular Adda riboswitch containing Tuner A. So we tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time. The following chart shows the dynamic curve measured every two hours. It can prove that Tuner A can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period.
Modular Btub riboswitch
By employing the Adda riboswitch, we found it’s possible to use our design principle to optimize the function of riboswitch. So we chose the Btub riboswitch, a repressing riboswitch which responds to adenosylcobalamin to verify the universal applicability of our guideline. After running our program, the first 150bp of BtuB, the original target gene of the Btub riboswitch was used to serve as Stabilizer. We introduced Tuner A to construct modular Btub riboswitch and sfGFP was reporter gene as the output of this system.
Then we proved the function of modular Btub riboswitch with the qualitative experiment. The fluorescence images can provide us with some useful information. It can result in a loss of ligand response when sfGFP was introduced directly after Btub riboswitch. Compared with the induced Btub fusion, the induced modular Btub riboswitch can show a greater induction difference, which demonstrates that Tuner has the ability to improve the function of riboswitch.
By microplate reader, we measured the intensity of sfGFP changing over time in our system. The following chart shows the dynamic curve measured every two hours. As we can see, the modular Btub riboswitch has a beautiful response curve.
Modular cobalamin riboswitch
Our team’s vision is a standardized and easily adaptable design principle to be used for riboswitch of different purposes. By referencing the previous iGEM project, we found that Paris_Bettencourt has created a cobalamin biosensor to measure vitamin B12. The cobalamin biosensor was based on a repressing riboswitch taken from a transcribed fragment upstream of a cobalamin biosynthesis gene, cbiB, which is found in Propionibacterium shermanii and has been demonstrated to be sensitive to vitamin B12. At first, they used EGFP as their reporter gene whose upstream is cobalamin riboswitch under control of the lac promoter. However, even in the absence of cobalamin, they had no GFP expression at all. Then they substituted EGFP with mRFP1 and inserted the first 24 bases of cbiB between them. They constructed a part consisting of the cobalamin riboswitch, the truncated cbiB gene, mRFP1 without start codon and rrnB T1 teminator. To test the functionality of this construct, we measured the fluorescence level emitted by recombinant strain in the presence of different concentration of adocobalamin by microplate reader. To our surprise, the intensity of mRFP1 was very low even without vitamin B12. The structure of RNAfold verified our prediction that Stabilizer they chose was too short that can't protect the structure of riboswitch.
With our principle, the cobalamin biosensor can be optimized. The modular cobalamin biosensor consisting of the repressing riboswitch, the first 81bp of the original target gene and Tuner A. To test the functionality of the improved construct, the modular riboswitch was under the lac promoter and controlled the expression of mRFP1. To test the functionality of the improved construct, we measured the fluorescence in the presence of increasing concentrations of adocobalamin by microplate reader. We can observe an obvious decrease in mRFP1 expression with the adocobalamin concentration increased, which demonstrates that the riboswitch was improved successfuly! The figure below selects the data when steady state is reached(at least two consecutive subsequent data points do not increase fluorscence).
Modular Four U riboswitch
After successfully constructing modular kinetic riboswitches, we then want to demonstrate that Tuner A can also apply to the thermodynamic riboswitch, which can response to temperature based on conformation changes. Therefore, we selected a thermodynamic switch named Four U that performed well and designed a modular Four U riboswitch containing the original switch, Stabilizer and Tuner A. Using docking matrix, we selected the first 132bp of RFP as Stabilizer because RFP can express under the control of Four U. The reporter gene was sfGFP and the construct were under the control of a constitutive promoter(BBa_J23119).
Given that the temperature threshold of Four U is 37℃, we set the culture temperature to 28℃, 37℃ and 42℃ in liquid LB medium respectively. After overnight culture, we choose 5 time points (8h, 24h, 30h, 36h, 48h) to measure fluorescence intensity of sfGFP protein by microplate reader. The results demonstrate that moduar FourU element is a useful thermodynamic riboswitch and works very well!
Summary
This year, we achieved a rational design principle of modular riboswitch. Many Tuners was utilized for tunable and efficient gene regulation. To verify the functionality of different Tuners, we engineered different modular Adda riboswitches including Tuner A to E respectively. All circuits selected sfGFP as the target gene. Using different Tuners, muti-level regulation can be achieved.
[http://2019.igem.org/Team:OUC-China/Model The method we used to design different Tuners is on this page!]
First, microplate reader was used to measure the fluorescence intensity of sfGFP. The figure below selects the data when steady state is reached(at least two consecutive subsequent data points do not increase fluorescence). The results demonstrate that these Tuners are capable of shifting and tuning the induction response of modular Adda riboswitches.
Then we also tested our modular Adda riboswitches by flow cytometer. The figure below shows the measured expression distributions at the same induction for modular Adda riboswitch with different Tuners.
By all the experiments mentioned before, we proved that Tuners work as expectations successfully. They are expected to serve as a powerful and tunable tool of riboswitch for future iGEM teams based on their demand.
If you are interested in the other parts we designed, you can click modular riboswitches containing Tuner B,Tuner C,Tuner D,Tuner Eand Tuner S.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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