Team:OUC-China/Design
Background
Our Pace
Adda Ribolego
Tuner
Stabilizer
GOI
Summarize Design Principle
More RiboLego
Btub Ribolego
Improved Cobalamin Riboswitch
Ribolego based on Four U
Antisense RNA
Future work
1. Background
Riboswitches have been discovered and characterized across numerous prokaryotes and eukaryotes. They are RNA parts that bind small molecules to regulate gene regulation. Riboswitches contain aptamer domain, comprising highly specific pockets in the 5’ untranslated region (5’-UTR) of the mRNAs that bind small molecules or ligands. Once a ligand selectively binds the aptamer, a conformational change in the RNA structure leads to a change in gene translation.
For synthetic biology application, riboswitches should be modular devices. However, the riboswitch’s specific RNA secondary structure is influenced not only by its own sequence, but also by the surrounding context including the proximal open reading frame (ORF) under the control of the riboswitch. Thus, substituting the original ORF with a new one can Interfere with its function, which strongly limits its applications scope. To overcome this lack of modularity, previous study created fusions comprised of a riboswitch, the first few hundred base pairs of the ORF was changed to a standard sequence, which named ‘Stabilizer’. A particular riboswitch and the sequence of the ‘Stabilizer’ are fixedly matched to insulate the riboswitch from the replaceable parts.
However, this approach fails in many circumstances as the N-terminal fused peptide translated from the ‘Stabilizer’ region may affect the folding and function of the desired protein, leading to unpredictable results. In 20XX, scientists invented a ‘Riboattenuator’ device which separates the translation of the ‘Stabilizer’ and the desired coding sequences (CDS) by a ribosome sliding-mediated chain triggered RBS exposure. So, the ‘Riboattenuator’ keeps the translational regulation ability of the riboswitch, but avoid the peptide translated from the ‘Stabilizer’ becoming the N-terminus of the desired protein.2. OUR PACE
The goal of our work was to propose a standardized design principle named “RiboLego”, based on the research of ‘Riboattenuator’ mechanism. We planned to make design and construction of modular riboswitch faster, easier and achieve more diverse regulation regulatory dynamics. A typical ‘Riboattenuator’ contains three functional elements, including the original riboswitch, ‘Stabilizer’ and ‘Tuner’ arranged from 5'to 3' end of the mRNA.
The ‘Stabilizer’ can protect the structure of riboswitch from interference. Two factors need to be considered when designing a ‘Stabilizer’, the source and length. There are many sources for users to choose, such as sequences from high-throughput screening methods, acquisition from the natural context of the riboswitch or an available sequence has been characterized in previous studies.
The ‘Tuner’ can couple the translation process of the ‘Stabilizer’ and the desired CDS, and prevent the translated ‘Stabilizer’ becoming the N-terminus of desired protein which may cause its unpredictable folding and dysfunction. By researching some key parameters of the ‘Tuner’, we will provide a series of ‘Tuners’ to regulate the response curve of riboswitch, achieving various dynamics.
To this end, we planned to designe an innovative software named RiboLego according to our model and experimental data, which could be used to design a modular riboswitch in the later stages based on the user's CDS sequence and desired 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 the Tuner
Adda riboswitch from Vibrio vulnificus is an riboswitch for translational activation which is 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 be translated. In Vibrio vulnificus, the gene which locates downstream of the Adda riboswitch is adenosine deaminase. To protect the structure of Adda riboswitch from being destroyed by GOI, we truncated the first 150bp of this gene as the ‘Stabilizer’ of modularized Adda riboswitch. Because our docking matrix suggested that a normal riboswitch structure would be observed when using 150bp as its length. Then a typical ‘Tuner’ was created and utilized to deal with the potential dysfunction of desired protein.
In Vibrio vulnificus, the gene which locates downstream of the Adda riboswitch is adenosine deaminase. To protect the structure of Adda riboswitch from being destoryed by GOI, we truncated the first 150bp of this gene as the 'Stabilizer' of modularized Adda riboswitch. Because our docking matrix suggested that a normal riboswitch structure would be observed when using 150bp as its length. Then a typical ‘Tuner’ was created and utilized to deal with the potential dysfunction of desired protein.
We defined a ‘Tuner’ element as a part including a repressing region, an RBS region and a coupled junction region. The repressing region is the reverse complement of a subsequence of the RBS region so that the ‘Tuner’ can form a hairpin with appropriate ΔG. The stop codon and start codon fused in the junction region. Ribosomes recruited by the upstream riboswitch can open up the hairpin of the ‘Tuner’ before dissociation at the stop codon in the junction region. Additional ribosomes can then assemble at the Tuner’s RBS and initiate translation at the first start codon of the introduced gene of interest. Therefore, the ‘Tuner’ can couple the riboswitch’s response and the translation of desired protein.
The superfolder green fluorescent protein (sfGFP) is the reporter gene to verify our modular Adda riboswitches, which is under control of the tetracycline promoter(Ptet). The results indicate that the protein dysfunction of the sfGFP reduced obviously.
3.1.2 More Tuners
In practical applications, the various application scenarios and associated systems may need different dynamic characteristics of the riboswitch. Obviously, the limited number of the wild type riboswitches cannot provide optional regulatory parts as a toolkit.
Talking with Professor Zhang Dawei 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 response curve with high output range may be more appropriate for the regulation of enzymes with low catalytic activity, the blue medium one may be more appropriate for layered regulatory networks that require a high output dynamic range. The red one with low output range may be more appropriate for the regulation of cytotoxic genes.
According to the mechanism of ‘Riboattenuator’, We realized that the ‘Tuner’ could be a perfect target to create the dynamics diversity. In order to meet this requirements, modeling help us to create more 'Tuners'. Five Tuners with various dynamic characteristics were selected as a part collection. According to the expression strength, we named Tuner A to E.
To demonstrate whether the ‘Tuners’ can make the response dynamics diverse, 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 results for more details!
3.2 Stabilizer
3.2.1 Source of Stabilizer
To stabilize the structure of riboswitch, many studies create a fixed sequence for a particular riboswitch in front of the GOI, which we named ‘Stabilizer’. Stabilizer can protect the structure of riboswitch from unpredictable interference by changeable downstream CDS. The source and length are two factors need to be considered when design the ‘Stabilizer’, as we mentioned above.
There are many ways to select the source of Stabilizer. However, we assumed that the natural context sequence of a particular riboswitch is the best sequence source for the ‘Stabilizer’, and this principle is universally applicable to various riboswitches.
In order to initially verify this idea, we utilized blast to catch the nature gene downstream of Adda riboswitch as the ‘Stabilizer’ 