Difference between revisions of "Part:BBa K3893010"

Revision as of 03:39, 22 October 2021

Up part of cassette for dsRNA constitutive production 2

Part of Modular Platform for dsRNA production.

dsRNA cassette production to improve stability

Upper part of the cassette for constitutive dsRNA production, which is composed of a terminator BBa_BBa_K3893017, promoter T7 BBa_I712074, RBS BBa_B0032 and GFP transcription unit that functions as a dropout BBa_K3893017.

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 SpeI site found at 164
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 918
Illegal SpeI site found at 164
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 158
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 164
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 164
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 834

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K3893010 short</partinfo>
+Part of Modular [https://2021.igem.org/Team:Ecuador/Part_Collection Platform for dsRNA production].
 dsRNA cassette production to improve stability
-Upper part of the cassette for constitutive dsRNA production, which is composed of a terminator (BBa_K3893017), promoter T7 (BBa_I712074), RBS (BBa_B0032) RBS.1 (BBa_K3893017) and GFP transcription unit that functions as a dropout (BBa_K3893017).
-Assembly system
+Upper part of the cassette for constitutive dsRNA production, which is composed of a terminator [https://parts.igem.org/Part:BBa_BBa_K3893017 BBa_BBa_K3893017], promoter T7 [https://parts.igem.org/Part:BBa_I712074 BBa_I712074], RBS [https://parts.igem.org/Part:BBa_B0032 BBa_B0032] and GFP transcription unit that functions as a dropout [https://parts.igem.org/Part:BBa_K3893017 BBa_K3893017].
+==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.
+* 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.
+* 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.
+* 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.
-) 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).
+. We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI.
-) We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.
+[[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]]