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

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

——Luis Serrano

By snapping together various pieces of different colors, shapes and sizes from a Lego box, a multitude of structures with different functions such as a boat, a car, and a building can be readily built. In the ideal world of synthetic biology, biological parts such as genes, promoters, and terminators are analogously treated as Lego blocks.

However, the key challenges in synthetic biology in real life exist on two main levels. One is the modularization and standardization of biological parts, while the other is the integration of these biological parts into devices with desired functions. Unlike Lego blocks, many of the existing parts are still incompatible and unpredictable, whose variability will crash the system sometimes.

So biologic parts of “Lego-ization” are necessary.

Since their discovery, riboswitches have been attractive tools in bacterial systems. Natural riboswitches are found with the highest frequency in the 5’-UTR of bacterial mRNAs, they have two main components: an “aptamer domain” and an “expression platform”. in response to the binding of a specific target molecule, they can regulate the expression of downstream genes through structural changes. Also, more artificial riboswitches are engineered to regulate the expression of proteins of interest.

The useful application of riboswitch

But due to context-dependent performance and limited dynamic range, the use of riboswitches is often restricted.

All in all, the three problems above make the riboswitch quite hard to design and employ to the application, which make it can’t be regarded as a modular device. Towards to the three problems, now the exiting strategies are describing following by:

~ To make the riboswitch as a modular plug-and-play device, scientists try to insert a sequence between the riboswitch and gene of interest to protect the structure of riboswitch from damage so that we can change the CDS easily. The sequence they have chosen is by random design and test by some high-throughput screening method such as SELEX.

~ To change the response functions of riboswitch, scientists try to design the expression platform by biology method and rational design. Many studies used the directed evolution to optimization the dynamics range of specific riboswitch and develop the bio-physics model to design it.

~To change the concentration of ligand in the experimental culture system, scientists often use the physics-based method, such as microfluidics device or replacing the media with fresh non-inducer-containing media .

By reviewing the exiting problem and solutions towards them, we are aware of some aspects worth optimizing. Adding a redundant sequence before the GOI directly will lead to the expression of fusion protein which may destroy the GOI's structure and function. And the method such as random design and directed evolution may waste too much time to achieve the goal you desired . While the effect of the microfluidics device is various between different labs.

This year, OUC-China proposed a standardized design principle named “RiboLego” which can break the deadlock we have mentioned before, making the riboswitch a modular, tunable one and easy to toggle between the on and off state. We hope our design will make it easier and more efficient for future igem teams to get the expected expression by using riboswitch .

We divide modular riboswitch into three parts: the original riboswitch, Stabilizer, Tuner from 5' to 3'.

Stabilizer is a sequence which can prevent the structure of the riboswitch from damage. It has a clear source to generate and the appropriate length designed by model.

Tuner placed between Stabilizer and the GOI to split them from each other has a function that reduces the expression probability of fusion protein and avoids destroying the GOI's structure and function. What's more, designed by model, Tuner can be used to control the riboswitch function precisely, achieving the desired level of expression.

We validate 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 including sfGFP, YFP, and mRFP1. The good results show the high universality of our design principle.

To toggle between the on and off state of kinetic switches, we use the model to design different asRNAs which target different region to activate or deactivate the riboswitch. We will optimize this system continuously and finally achieved to regulate the on-off state of riboswitch.

All in all, inspired by the three existing blocks, we design our alternative riboswitch design frameworks, 'RiboLego', to make the riboswitch modular, tunable, reliable and time-saving.

Click here to get more information about our achievements!