Difference between revisions of "Part:BBa K3893015"

Revision as of 04:24, 18 December 2021

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, RBS (BBa_B0034), promoter T7 (BBa_I712074) and terminator (BBa_K3893007).

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
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 798
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 44
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 798
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 798
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 128

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3893015 short</partinfo>
-Part of Modular Platform for dsRNA production
+Ppart of the [https://2021.igem.org/Team:Ecuador/Part_Collection Modular Platform for dsRNA production] in the proyect [https://2021.igem.org/Team:Ecuador/project AgroBactory 593].
 Lower part of the cassette for constitutive dsRNA production, which is composed of a GFP transcription unit that functions as a dropout, RBS (BBa_B0034), promoter T7 (BBa_I712074) and terminator (BBa_K3893007).
+==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]]