RNA

Part:BBa_K3893003

Designed by: Carolina Panchana   Group: iGEM21_Ecuador   (2021-10-20)


dsRNA designed for the target gene SGE1

This sequence is part of the Modular Platform for dsRNA production in the proyect AgroBactory 593.

dsRNA designed to silence the SGE1 target gene via RNAi mechanism.

Usage and Biology

Once inside the pathogen, this dsRNA is further processed into RNA interference that can regulate the expression of any gene in the genome. SGE1 promotes the expression of SIX, which is required for the formation of conidia. This has been a target gene to apply dsRNA against FOC R1 [1].

Assembly system

Before constructing the modified plasmids and using them, we took into account the following:

  • Have a Biobrick-compatible backbone, in case you don't have it you could modify it with PCR overhang and add RFC10 prefix and suffix.
  • The following restriction enzymes are used: EcoRI, PstI, SpeI, XbaI, BglII and KpnI. Therefore the dsRNA sequence should not contain these restriction sites.
  • Previously carry out PCR overhang to the dsRNA sequence to add the restriction sites: BglII and KpnI.

Once the above considerations have been met, the following assemblies are performed:

1. We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI.

domestication.
Step 1

2. Assembly 3A adapted: We digested the UP plasmid with EcoRI - SpeI, the DP plasmid with XbaI - PstI and a Biobrick compatible backbone with EcoRI - PstI. Then we ligated and obtained as a product the modified plasmid (dsRNA receptor).

domestication.
Step 2

3. We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.

domestication.
Step 3


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 438
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 438
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 438
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 438
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] V. Gurdaswani, S. B. Ghag, and T. R. Ganapathi, “FocSge1 in Fusarium oxysporum f. sp. cubense race 1 is essential for full virulence,” BMC Microbiology, vol. 20, no. 1, p. 255, Aug. 2020, doi: 10.1186/s12866-020-01936-y.


[edit]
Categories
Parameters
None