Part:BBa_K2615009

The miniToe family test system No.2.

Background of 2018 OUC-China' project——miniToe family

This year, we design a toolkit named miniToe family focused on translational regulation, which is composed of a RNA endoribonuclease, Csy4 and a RNA module (hairpin). In our project, the cleavage function of Csy4 releases a cis-repressive RNA module (crRNA, paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.

Fig.1 Csy4 and hairpin can form a stable structure.

Fig.2 The structure of wild type hairpin.

We further design four Csy4 mutants by point mutation, Csy4-Y176F, Csy4-H29A, Csy4-F155A and Csy4-Q104A. At the same time, we redesign 5 different hairpin mutants named miniToe(5 different types of Csy4 recognition sequence), miniToe-1, miniToe-2, miniToe-3, miniToe-4, miniToe-5 and miniToe-WT.

The function of miniToe

As an exchangeable module, the crRNA-RBS duplex was 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. The miniToe-2 is one of the miniToe family which we design to meet the aim, "One system, diverse expression".

The No.2 test system for miniToe family

This part is called miniToe test system-2. It is composed by four basic parts. A constitutive promoter J23119 is used to drive the expression of sfGFP. A miniToe-2 element is placed before sfGFP coding sequence containing a complementary sequence and a Csy4 recognition sequence. The RiboJ module is inserted between crRNA element and the J23119 promoter. RNA parts often serve as critical components in genetic engineering.

The test system for miniToe family consists of miniToe test system-1, miniToe test system-2, miniToe test system-3, miniToe test system-4, and miniToe test system-5. The length of each part is 901bp, and the electrophoretic result shows the validity of stripe.

Fig.3 The electrophoretic result of miniToe family test system.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 197