Difference between revisions of "Part:BBa K3893002"

Revision as of 04:06, 22 October 2021

dsRNA designed for the target gene ERG11 Part of Modular Platform for dsRNA production. Description: dsRNA designed for the target gene ERG11

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K3893002 short</partinfo>
+Part of Modular [https://2021.igem.org/Team:Ecuador/Part_Collection Platform for dsRNA production].
 Description: dsRNA designed for the target gene ERG11
+==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:
+. We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI.
+[[File:T--Ecuador--PartColl_Assembly1.png|400px|thumb|center|alt=domestication.|Step 1]]
+. 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).
+[[File:T--Ecuador--PartColl_Assembly2.png|800px|thumb|center|alt=domestication.|Step 2]]
+. We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.
+[[File:T--Ecuador--PartColl_Assembly3.png|600px|thumb|center|alt=domestication.|Step 3]]
 <!-- Add more about the biology of this part here