Part:BBa_K3893004

dsRNA designed for the target gene SIX1

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

dsRNA designed to silence the SIX1 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. SIX1 plays the role of a virulence factor This has been a target gene to apply dsRNA against Foc R4T [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.

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).

Step 2

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

Step 3

References

[1] S. Widinugraheni et al., “A SIX1 homolog in Fusarium oxysporum f.sp. cubense tropical race 4 contributes to virulence towards Cavendish banana,” PLOS ONE, vol. 13, no. 10, p. e0205896, Oct. 2018, doi: 10.1371/journal.pone.0205896.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 369
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 288