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

Inspiration

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 syst1em 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

①Metabolism and behavioural regulation

Directing mobility of bacteria to specific locations using theophylline in a ligand‐dependent manner.

②Screening for traits

Lysine riboswitch control antibiotic resistance to screen for Escherichia coli strains with higher lysine fermentation efficiency.

③Regulation of genes

Use Aminoglycoside riboswitch to increase bacteria resistance to antibiotics.

④Production of compounds

RNAT (temperature response) can rapid response to temperature for the production of compounds without the use of costly ligands.

⑤Biosensor

Flavonoid riboswitch can detect the flavonoid contamination.

⑥Bioremediation

ykkC riboswitch is able to respond to the environmental toxin guanidine to break it down.

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

Non-plug and play device Hard to control its response function Impossible to reset its state

Team Paris_Bettencourt has used a riboswitch whose ligand is vitamin B12 to measure the concentration of vitamin B12 in foods. They directly added eGFP after the riboswitch but found that no eGFP expression at all. After that, although eGFP was substituted with mRFP1 and natural truncated protein was inserted between mRFP1 and the riboswitch, they finally observed a bad result. We guess that the structure of riboswitch has been changed. Natural riboswitches primarily serve as key autonomous regulators of diverse metabolic processes. However, the application of riboswitch was restricted by the low dynamic regulatory ranges and low tunability. To effectively regulate genes with riboswitch, an appropriate strategy must be employed to tune the response curve, achieving the multi output by responsing to single input. Because small molecules are often hard to degrade in the experimental culture system, it often causes some problems and makes the system away from prediction. 2018 Team William and Mary also focused on this problem. By talking with Prof. Wang, we found the kinetic switch could not easily toggle between the on and off state, which results in some logic functions is lost.

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!