Part:BBa_K2615026

The miniToe motility detection system.

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.

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

The structure of miniToe

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. We design the translational activator with three modular parts:

• a crRNA to serve as translation suppressor by paring with RBS
• a Csy4 site as linker between crRNA and RBS
• a CRISPR endoribonuclease Csy4

Fig.2 The structure of wild type hairpin.

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miniToe-motA

It is composed by four basic parts. A constitutive promoter J23119 is used to drive the expression of following interesting gene. A crRNA 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 whole structure can be recognized by Csy4, and different miniToes will have totally different recognition rate. 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 motA. This is a real-world application of miniToe family. </p>

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 13
  Illegal NheI site found at 36
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1
  Illegal BglII site found at 513
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 251
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1019