Part:BBa_K3893015
Down part of cassette for dsRNA constitutive production 4
Ppart of the Modular Platform for dsRNA production in the proyect AgroBactory 593.
Lower part of the cassette for constitutive dsRNA production, which is composed of a GFP transcription unit that functions as a dropout (BBa_K3893018), Shine Dalgarno (BBa_K3893021), RBS (BBa_K3893022), 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 a 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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 798
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 44
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 798
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 798
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 128
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