Part:BBa_K2615019

MiniToe polycistron B: a new system using miniToe.

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 design miniToe family to meet the aim, "One system, diverse expression". This means by using one system we can even achieve flexable expression of target gene. So 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).

miniToe polycistron

Having designed different hairpin structures and finished experimental verification, we think it is a good idea to apply it to the polycistronic to achieve more functions by using miniToe. The polycistronic structure can meet the requirements of simultaneous and large-scale expression of different genes. We insert the hairpin structure(miniToe) into the polycistron and apply the interaction between the hairpin structure and the Csy4 enzyme to achieve the regulation of different ratio of multiple gene expression levels, meeting the requirements of practical application.

Fig.2 The working process of miniToe polycistron.

From now, we construct two polycistron(miniToe polycistron A and miniToe polycistron B) by inserting three miniToes before, between and behind two CDS. We use miniToe-WT(BBa_K2615020) before and behind miniToe polycistron B(BBa_K2615018), meanwhile the miniToe-6(BBa_K2615017) is used between two CDS. And we use sfGFP and mCherry to test our system. The result shows we can totally change the origin ratio of two genes.

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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 197