Difference between revisions of "Part:BBa K3893009"

Latest revision as of 03:59, 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 terminator BBa_K3893006, promoter T7 BBa_K3893020, RBS BBa_K3893019 and GFP transcription unit that functions as a dropout BBa_K3893018.

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
COMPATIBLE WITH RFC[12]
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.rc site found at 698

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3893009 short</partinfo>
-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_B0034), promoter T7 (BBa_I712074) and terminator (BBa_K3893007).
+Part of Modular [https://2021.igem.org/Team:Ecuador/Part_Collection Platform for dsRNA production].
-Assembly system
+Lower part  of the cassette for constitutive dsRNA production, which is composed of a terminator [https://parts.igem.org/Part:BBa_K3893006 BBa_K3893006], promoter T7 [https://parts.igem.org/Part:BBa_K3893020 BBa_K3893020], RBS [https://parts.igem.org/Part:BBa_K3893019 BBa_K3893019] and GFP transcription unit that functions as a dropout [https://parts.igem.org/Part:BBa_K3893018 BBa_K3893018].
+==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]]