Difference between revisions of "Team:OUC-China/Demonstrate"

1. Background

Riboswitches have been discovered and characterized across numerous prokaryotes and eukaryotes. They are RNAs that bind small molecules to regulate metabolism and gene regulation. Riboswitches contain aptamer domain sites, comprising highly specific pockets in the 5’ untranslated region (UTR) of the mRNAs that bind small molecules or ligands. Once a ligand selectively binds an aptamer site, a conformational change in the RNA structure leads to a change in gene expression.

To be truly useful for synthetic biology, riboswitches should be modular “plug and play” devices. However, the riboswitch’s specific RNA secondary structure is influenced not only by its own sequence, but also by the surrounding genetic context including the proximal open reading frame (ORF) under the control of the riboswitch. Thus, substituting the original ORF with a new one can nullify the desired riboswitch response to a given ligand, so GOI can’t express at all, which strongly liits its applicatios scope. To overcome this lack of modularity, many studies have created fusions comprised of a riboswitch, the first few hundred base pairs of its working ORF, which we name “Stabilizer”, and a gene of interest. By introducing Stabilizer, the riboswitch can respond to the ligand and GOI can express as fusion protein. However, this approach fails in many circumstances as it may alter the gene’s structure and functionality, leading to unpredictable results.

2. OUR PACE

①Natural riboswitches are found with the highest frequency in the 5’-UTR of bacterial mRNAs, where they regulate the expression of downstream genes through structural changes undergone in response to the binding of a specific target molecule.

②When GOI located downstream of the riboswitch changes, the structure of the riboswitch will be destroyed. Then it can’t respond to the ligand. So the GOI can’t express at all.

③By introducing stabilizer, the riboswitch can respond to the ligand but the redundant sequences may influence the structure and function of GOI.

④Using tuner, we can tackle this problem. The tuner can reduce the expression probability of fusion protein and allow for predictable tuning. More tuners are designed to make diverse expression level.

⑤We explore the resource and length of stabilizer to propose the design principle of stabilizer.

⑥We choose different riboswitches to express all kinds of GOI. By doing this, we can verify the design principle of modular riboswitch is universal.

⑦Because ligands are difficult to degrade, the on-off state of riboswitch is not quickly regulated at present. We design asRNA to solve this problem.

The goal of our work was to propose a standardized design principle named “RiboLego”, making construction of modular riboswitch faster, easier, more stable and achieve more diverse regulation. Modular riboswitch we defined contains three functional elements, including the original riboswitch, Stabilizer and Tuner from 5’ to 3’.

图

Stabilizer can protect the structure of riboswitch from damage. Two factors need to be considered when designing Stabilizer, the source and length. There are many sources for users to choose, such as high-throughput screening methods, acquisition from the original genome and the working gene in the past study. As for its length, dry group members help us determine it by docking matrix and RNAfold.

Tuner can reduce the expression probability of fusion protein and make improvement of riboswitch function. We provided a series of Tuners to regulate the response curve of riboswitch, achieving multiple output.

To this end, we designed an innovative software named RiboLego which will provide a modular riboswitch in the later stages based on the user's target sequence and expected expression level.

In order to reach our ambitious goal, we employed Adda riboswitch to demonstrate the usability of design principle. Based on this, more riboswitches are changed into RiboLego to indicate the universal of our guideline.

3. RiboLego based on Adda

3.1 Tuner

3.1.1 The structure of Tuner

Adda riboswitch from Vibrio vulnificus is an activating riboswitch responsive to 2-aminopurine. When 2-aminopurine exists, it can bind the aptamer domain of riboswitch, causing a structural rearrangement which can open up RBS, so GOI can translate.

In Vibrio vulnificus, the gene which locates downstream of the Adda riboswitch is adenosine deaminase. To protect the structure of Adda riboswitch from destorying by GOI, we truncated the first 150bp of this gene as Stabilizer of modular Adda riboswitch. Because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. If you interested in how to design the Stabilizer, you can click here. Then Tuner was created and utilized to deal with the fusion protein phenomenon.

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 help GOI express normally and facilitate tuning of a riboswitch’s response .

The superfolder green fluorescent protein (sfGFP) is the reporter gene to verify our modular Adda riboswitches, which is under control of the tetracycline promoter.The result indicate that the expression probability of fusion protein reduce obviously. You can get more information in result.

3.1.2 More Tuners

In many practical applications,the riboswitch response curve is restricted by application category and associated system. Depending on these restrictions, proper tuning of riboswitches acting as autonomous control systems may require minimization of basal levels, operation across higher expression levels, or maximization of the change in expression levels. Obviously, the original riboswitch function is single and it cannot achieve multi-level regulation.

Talking with Professor Zhang in the 5th Synthetic Biology Young Scholar Forum, we found it was necessary to make the function curve of riboswitch diverse. For example, in different environments, the same riboswitch is required to have different response ranges. The yellow high level response curve may be more appropriate for the regulation of enzymes with low catalytic activity, the blue medium one may be more appropriate for regulatory networks that require a large change in protein levels and the red low one may be more appropriate for the regulation of cytotoxic genes.

In order to meet this requirements, modeling help us to create more Tuners. Five Tuners were selected as a part collection. According to the expression strength, we named Tuner A to E.

To demonstrate whether Tuners can shift and optimize the response curve of riboswitch, we created five modular Adda riboswitches by combining the original Adda riboswitch, Stabilizer and five Tuners respectively!

Wet experiments show that our system can work well! Click result for more details!

结果图(5个tuner单浓度结果图)

3.2 Stabilizer

3.2.1 Source of Stabilizer

Stabilizer can protect the structure of riboswitch from damage. Two factors need to be considered when designing Stabilizer, the source and length. There are many sources for users to choose, such as high-throughput screening methods, acquisition from the original genome and the working gene in the past study. As for its length, dry group members help us determine it by docking matrix and RNAfold.

To stabilize the structure of riboswitch, many studies create and insert a sequence in front of the GOI, which we named “stabilizer”. There are many ways to select the source of Stabilizer, such as high-throughput screening methods, acquisition from the original genome and the working gene from paper. When testing Tuners, we utilized blast to catch the nature gene downstream of Adda riboswitch as Stabilizer. Furthermore, in order to verify the its source is changable, we also chose GFP as Stabilizer of Adda riboswitch because Adda riboswitch can express GFP directly in the past study.

By expriments, we can verify that the source of Stabilizer is diverse.

3.2.2 Length of stabilizer

Stabilizer should be long enough to maintain the secondary structure of most riboswitches but short enough to minimise the overall size of the system.So we explored the length of Stabilizer.

According to using docking matrix, we can get Stabilizer of appropriate length. Detailed methods can be referred to modeling and software.

In order to verify our software, we chose two good Stabilizers which are able to prevent the structure from destroying and two bad Stabilizers whose length were too short to maintain the structure.

The results show that our software was useful and reliable!

3.3 GOI

To ensure that our modular riboswitch will work with a variety of different proteins, we substituted sfGFP with EYFP. Using the new interest gene, we tested the effect of modular Adda riboswitch consisting the original Adda riboswitch, STA150 and Tuner A. The result verified that the target gene is indeed replaceable, the modular riboswitch secondary structure is not affected!

3.4 The design principle of modular riboswitch

Fortunately! So far, we have been able to summarize a complete set of modular riboswitch design principles: modular riboswitch consisting of original riboswitch, stabilizer, and tuner from 5' to 3'.

We have applied this design principle to Adda riboswitch. The experience verified that GOI is replaceable and the modular riboswitch can regulate the expression of GOI. Further, we will use this design principle to create more RiboLego!

4. More RiboLego

4.1 RiboLego based on Btub

After introducing an activating riboswitch, a repressing riboswitch is expected to employ to valiate our design principle. In absence of ligand, the repressing riboswitch can expose RBS, making GOI can express. When ligand exists, GOI can’t express at all. Taking it into consideration, we employed Btub riboswitch responsive to VB12 from E.coli. By our program, the first 150bp of BtuB, the original target gene of the Btub riboswitch was used to serve as Stabilizer. We selected Tuner A and E to design two modular Btub riboswitches and used sfGFP as the reporter gene to test whether the expressions of these two systems are as expected.

Although introducing the stabilizer can protect the structure of the riboswitch from destroying, we worried that its accumulation can lead to increased metabolic pressure on cells, affecting cell function and the expression of target genes. Therefore we decided to design a Tuner S containing ssrA protein degradation tag to degrade Stabilizer.

The results show that Stabilizer and Tuner constructed on Btub riboswitch can work well!

4.2 IMPROVE: RiboLego based on cobalamin Riboswitch

By referencing the previous iGEM project, we found that Paris_Bettencourt has created a cobalamin biosensor to measure vitamin B12. The cobalamin biosensor is based on a 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 B12. At first, they used EGFP as their reporter gene, 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, the result was bad, too..

In order to verify the universality of our modular riboswitch, we improved part号. Using our software, we selected the first 81bp of cbiB as the stabilizer and used tuner A to control the expression of mRFP. The results proved that we successfully designed the modular cobalamin biosensor by our design principle!

4.3 IMPROVE: RiboLego based on Four U

Riboswitches can furthermore be classifified into thermodynamic and kinetic switches. Different from kinetic switches, thermodynamic switches can reversibly and repeatedly toggle between on- and off-states, depending on temperature. Thermodynamic switches are temperature-sensing RNA sequences in 5’UTR of their mRNAs. At low temperature, they can fold into the structure, blocking access of ribosome; at high temperature, it open conformation, increasing the efficiency of translation initiation.

After successfully creating modular riboswitch with our method on kinetic switches and demonstrate that Tuner can control the expression of a range of functional outputs, we started to build modular thermodynamic riboswitch, and here we made part BBa_K115002( Four U) improvement based on TUDelft team in 2008.

Four U is an RNA thermometer that can be used for temperature sensitive post-transcriptional regulation that initiates translation at 37°C. Fortunately, we found that OUC-China team had used Four U to successfully express RFP in 2005, which provided us with great convenience! So we selected the first 132bp of RFP as Stabilizer of Four U. Then we used Tuner A to change the exprssion level. The superfolder green fluorescent protein (sfGFP) is the reporter gene to verify our modular Adda riboswitches.

Experimental results show that we have successfully constructed a modular thermodynamic riboswitch and changed its response curve.

5. AsRNA

Thermodynamic switches are found in energetic equilibrium between their on- and off-state. If switching is triggered, the equilibrium distribution

shifts towards the new energetically best conformation. This implies that thermodynamic switches can reversibly and repeatedly toggle between on- and off-states. In contrast, kinetic switches are trapped in one state, depending on whether the ligand was present at the time of folding. Because ligands are hard to degrade, the state of the dynamic riboswitch is difficult to change.

With the help of Professor Li, we finally untilized antisense RNA to tackle this problem. Antisense RNA is endogenous in E. coli that do not require heterologous proteins to function. Owing to its simple design principles, small size, and highly orthogonal behavior, the engineered genetic parts has been incorporated into genetic circuits. Antisense RNA can be thought to consist of two regions: a target binding region (TBR) containing a sequence that is complementary to the target gene, and an Hfq binding site which allows for binding of the Hfq protein. Hfq is a native chaperone protein that mediates RNA?RNA interactions by binding to a particular RNA binding site on the asRNA molecule. In our work, the engineered MicF binding site (MicF M7.4) was used as Hfq binding site because it performed well with low off-target effect in previous studies.

By using model to change TBR, we hope to utilize asRNA to change the on-off state of riboswitch.

When targeting RBS of Adda riboswitch, asRNA can close modular Adda riboswitch even in presence of 2-AP. At the same time, when targeting RBS of Tuner E, asRNA is able to open modular Btub riboswitch even in presence of VB12.

The result show that we've been able to regulate the on-off state of riboswitch!

6. In the future

This year, OUC-China proposed the design principle of modular riboswitch consisting of original riboswitch, Stabilizer, and Tuner from 5' to 3', and used it to create “RiboLego”. Besides, we introduce asRNA so that gene expression in engineered systems can be more easily regulated.

In the future, there are some works to improve our project. Firstly, we want to create more Tuners based on this design principle to achieve more level regulation in different environments. Secondly,We expect our software can help future iGEM teams to easily use riboswitches to express anticipated GOI and get the ideal level of expression. Finally, we hope to use the cell-free system to optimize RiboLego from the perspective of the department of engineering, and use vesicles to wrap asRNA so that it can carry out accurate real-time regulation of riboswitch state for many times.