Device
miniToe mo

Part:BBa_K2615026

Designed by: Yunqian Zhang   Group: iGEM18_OUC-China   (2018-09-10)


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.

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 motA 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.

Fig.3 The working process of miniToe-motA.

Result

Proof of functions about the miniToe motility detection system.

Five groups have been set, a test group and four control groups. And the results shown below have proved that our system can work as expectation.

The control groups A and B including positive group and negative group. Plates were inoculated with E.coli RP437 (A1, A2, A3) that have motility and they can move arbitrarily in the plates. The plates on right are ΔmotA strains(the motA-deletion strain) (B1, B2, B3), E.coli RP6666, which have no motility so the strains stay on the center. We have three biological replicates in this experiment.

Fig.4-1 The result of control groups E.coli RP437 and RP6666.

The test group C. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe structure).Without the gene motA, the E.coli cannot move. And the Csy4 have no big influence on strain compared with the ΔmotA strain. The little round of papers indicates the places of inducer IPTG (Isopropyl β D thiogalactopy ranoside). We have three biological replicates in the experiment.

Fig.4-2 The result of test group Csy4-ΔmotA.


The test group D. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe structure but no Csy4. The circuit is on the control of miniToe and its downstream gene motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.

Fig.4-3 The result of test group miniToe-motA.


The test group E. The strain we culture in plates is miniToe-motA with Csy4. The strain have the whole miniToe system which means motA can be regulated by miniToe. In the picture, the E. coli move everywhere in the plates, proving that with the regulation of miniToe and Csy4, the downstream gene motA come into play. The E. coli can move everywhere in the plate.

Fig.4-4 The result of test group miniToe-motA with Csy4.


In summary

As we can see, test group strains can move everywhere in the plate and the control groups strains can not move.The test group work as expectation compared to the control groups. But there is no time for us to test more miniToe mutants and Csy4 mutants in miniToe family. We want to realize the function of regulation by using different miniToe family members in the future. So we still have a lot of work to do.


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


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