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

We are still like the Wright Brothers, putting pieces of wood and paper together.

——Luis Serrano

Research Background

One of the key challenges in synthetic biology is how to improve the modularity of genetic parts and circuits. Unlike Lego blocks, many of the existing parts are still incompatible, whose unpredictable function or dynamic behavior will destroy the desired biological function we designed sometimes.

Since it was discovered, riboswitch has been an attractive tool in bacteria. Natural riboswitches are found with the highest frequency in the 5'-UTR of bacterial mRNAs. By responding to the specific trigger molecule, they could regulate the translation of downstream CDS through secondary structural change. Also, more and more artificial riboswitches are engineered to regulate the translation of the proteins of interest.

The useful application of riboswitch

However, due to context-dependent performance and limited dynamic range, the usage scenario of riboswitches is still restricted. The necessary secondary structures of the riboswitches are generally sensitive to its context sequence. When combining a riboswitch with various downstream genes, unpredictable interaction between the riboswitch and the adjacent CDS and RNA secondary structure formation may interfere with its normal function seriously.

Above problems make the riboswitch hard to be designed and employed for application, which also makes it cannot be regarded as a well-modularized device. Towards to this unsatisfactory situation, the exiting strategies provided by previous researches are described as follows:

~ To make the riboswitch as a modular plug-and-play device, scientists tried to insert a ‘insulator’ sequence between the riboswitch and gene of interest to protect the structure of riboswitch from interference, so that the CDS could be changed in a modularized way. The sequence they chose is from sequence selection by some high-throughput screening method such as SELEX. In 2017, an Ribo-attenuator device was achieved to solve above problem in E. coli. By designing a chain triggered translation mechanism, the redundant peptide translated from the ' insulator ' could be decoupled with the desired protein.

~ To optimize the response of riboswitch, scientists tried to design the riboswitches by evolutionary strategy. Many studies employed the directed evolution to optimize the dynamic range of specific riboswitch and developed the bio-physics model to design it.

~ To turn off the riboswitch from its functional state, scientists often use the physics-based method to remove the corresponding ligand from the culture system, which is impractical in many industrial application scenarios. Until today, there is no an ideal way to turn off the riboswitch without replacing the medium.

Our Research

By reviewing the exiting problems and solutions towards them, we are aware of some aspects are worth being researched and optimized. For the first item above, although the strategy of the ‘Ribo-attenuator’ has been proved as a feasible solution, many key general design principles are still unclear. We systematically researched the sequence source & length of the ‘stabilizer’, structure of the ‘tuner’ (the interaction relationship between the upstream stop codon and the downstream start codon), which helped us set up the complete design principles and models to design such Ribo-attenuator devices (named as RiboLego).

For the second item, the method such as directed evolution may waste too much time to optimize the dynamic range of riboswitch. The design rules of the ‘tuner’ part were described in our work, and the RiboLegos with various dynamic ranges were realized by using our design rules, which brought the optimization mode of riboswitches into the realm of rational design.

For the third item, we developed a method to turn off the riboswitch under ligand existence without medium replacement, which will extend the application scenarios of riboswitch to actual industrial field.

Summary

This year, OUC-China proposed a standardized design principle named “RiboLego” which can break the deadlock we have mentioned before, making the riboswitch as a modular, tunable genetic device and easy to toggle between the ‘On’ and ‘Off’ state. We hope our design principles and models will make it easier and more efficient for synthetic biologists and future iGEM teams to get the predictable results during using riboswitch.

We divided modular riboswitch into three parts as the previous attenuator design:the original riboswitch, ‘Stabilizer’ and ‘Tuner’ arranged from 5' to 3' direction of mRNA.

Stabilizer is a sequence which can work as an insulator to prevent the interference between riboswitch and downstream CDS. We figure out the principle of the sequence design and the appropriate length designed by modeling.

Tuner placed between the ‘Stabilizer’ and the gene of interest(GOI) to work as a chain trigger to couple the translation of the ‘Stabilizer’ and that of downstream CDS. This reduces the probability of folding abnormality of the desired protein, which caused by the N-terminal fused peptide translated from the ‘Stabilizer’. What's more, designed by model, the ‘Tuner’ could be used to control the riboswitch function precisely, for achieving the desired level of expression.

We validated our design principle in different riboswitches including three kinetic switches: Adda riboswitch, Btub riboswitch, cobalamin biosensor, and one thermodynamic switch: Four U riboswitch. What's more, three different kinds of GOI is used: sfGFP, YFP, and mRFP1. The results show the high modularity and universality of the RiboLegos.

To control the riboswitches without medium replacement for removing the inducer, we used the model to design different asRNAs which target different regions to activate or deactivate the riboswitch. We plan to optimize this system continuously and finally achieved to regulate the on-off state of riboswitch.

All in all, inspired by the previous attenuator and its mechanism, we designed our alternative riboswitch design frameworks, 'RiboLego', to make the riboswitch modular, tunable, reliable.

Click here to get more information about our achievements!

REFERENCE

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[3] https://2015.igem.org/Team:Paris_Bettencourt/Project/VitaminB12

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[9] Lynch S A , Desai S K , Sajja H K , et al. A High-Throughput Screen for Synthetic Riboswitches Reveals Mechanistic Insights into Their Function[J]. Chemistry & Biology (Cambridge), 2007, 14(2):173-184.

[10] Hoynes-O’Connor, Allison, Moon T S . Development of Design Rules for Reliable Antisense RNA Behavior in\r, E. coli[J]. ACS Synthetic Biology, 2016:acssynbio.6b00036.

[11] Lee Y J , Kim S J , Amrofell M B , et al. Establishing a multivariate model for predictable antisense RNA-mediated repression[J]. ACS Synthetic Biology, 2018.

[12] Lee Y J , Moon T S . Design rules of synthetic non-coding RNAs in bacteria[J]. Methods, 2018:S1046202317303389.