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MiniToe, a cis-regulatory RNA element

The structure of miniToe

This year, we design a new cis-regulatory RNA element named miniToe which can be recognized by Csy4. The translational control module is constructed by inserting a Csy4 recognition site between a RBS and cis-repressive RNA element, which can be specifically cleaved upon Csy4 expression. The whole system including miniToe and Csy4 is a kind of translational control module miniToe.
The translational activator with three modular parts:

• A cis-repressive RNA (crRNA) served as a translational suppressor by pairing with RBS as the critical part of miniToe structure.
• A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
• A CRISPR endoribonuclease Csy4.
  

Fig.1 The structure of miniToe on the level of RNA.

The function of miniToe

As an exchangeable module, the crRNA-RBS duplex is forwardly and reversely engineered to modulate the dynamic range of translational activity. Upon recognition by Csy4, the RNA can be cleaved after a specific nucleotide within the Csy4 site, and the piece of the crRNA element will be released from the masked SD sequence, thus endowing the programming of gene expression in the translation level with higher feasibility. By constructing this combination, we make it possible to regulate the expression of downstream gene sfGFP in order to test our miniToe family.

Fig.2 The process of miniToe's function.

The aim of miniToe family

We design miniToe family to meet the aim, "One system, diverse expression". That means by using one system we can even achieve flexable expression of target gene. The miniToe-WT which is a wild type hairpin is one of the miniToe family. And then we use sfGFP as our reporter.( miniToe test system-1, miniToe test system-2, miniToe test system-3, miniToe test system-4, and miniToe test system-5)

By quantitatively adjusting the miniToe hairpin, we can even achieve a stoichiometry expression of the target proteins in a polycistron gene expression construct. In our design, the duplex can be modularized, the mutants of the crRNA may potentially fine-tune the translational activator. Thus offering diversity to future design and integration into complex genetic circuits.

Result

To explore the feasibility and function of miniToe, we designed the circuit below as our test system in order to test the function of miniToe structure. We use Ptac as the inducible promoter of Csy4 to control the existence of Csy4 or not. At the same time, we construct the miniToe before the sfGFP which is a symbol of target gene in our circuit. And this circuit is controlled by a constitutive promoter form Anderson family named J23119.

Fig.3 The two plasmids of miniToe test system.

Without Csy4, the crRNA pairs with RBS very well, so the switch just turns off, which means that no protein will be produced. Otherwise, with the presence of Csy4, the translation turns on. In this way, the expression of downstream gene can be regulated.
By experiments we have proved that our system can work well!

Fig.4 The result of our first system.

[http://2018.igem.org/Team:OUC-China/Results Click here for more details!]

Sequence and Features