Difference between revisions of "Part:BBa K3893002"

Latest revision as of 03:48, 18 December 2021

dsRNA designed for the target gene ERG11

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

dsRNA designed to silence the ERG11 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. ERG11 plays a role in the synthesis of ergosterol as well as conidia formation. 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

Sequence and Features

References

[1] T. Dou et al., “Host‐induced gene silencing of Foc TR4 ERG6/11 genes exhibits superior resistance to Fusarium wilt of banana,” Plant Biotechnol J, vol. 18, no. 1, pp. 11–13, Jan. 2020, doi: 10.1111/pbi.13204.