Difference between revisions of "Part:BBa K3893009"

Revision as of 01:33, 22 October 2021

Down part of cassette for dsRNA constitutive production 1

Part of Modular Platform for dsRNA production

Lower part of the cassette for constitutive dsRNA production, which is composed of a GFP transcription unit that functions as a dropout, RBS (BBa_K3893019), promoter T7 (BBa_K3893020) and terminator (BBa_K3893006).

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. 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 product the modified plasmid (dsRNA receptor). 3) We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.

Sequence and Features