Difference between revisions of "Part:BBa K3893010"
 
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<partinfo>BBa_K3893010 short</partinfo>
 
<partinfo>BBa_K3893010 short</partinfo>
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Part of Modular [https://2021.igem.org/Team:Ecuador/Part_Collection Platform for dsRNA production].
  
 
dsRNA cassette production to improve stability
 
dsRNA cassette production to improve stability
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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).
 
  
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Assembly system
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Upper part of the cassette for constitutive dsRNA production, which is composed of a terminator [https://parts.igem.org/Part:BBa_BBa_K3893007 BBa_BBa_K3893007], 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].
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==Assembly system==
  
 
Before constructing the modified plasmids and using them, we took into account the following:
 
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.
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* 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.
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○ The following restriction enzymes are used: EcoRI, PstI, SpeI, XbaI, BglII and KpnI. Therefore the dsRNA sequence should not contain these restriction sites.
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* 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:
 
Once the above considerations have been met, the following assemblies are performed:
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1) We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI.
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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 product the modified plasmid (dsRNA receptor).
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1. We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI.
−
3) 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]]
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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).
 +
 
 +
[[File:T--Ecuador--PartColl_Assembly2.png|800px|thumb|center|alt=domestication.|Step 2]]
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3. We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.
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[[File:T--Ecuador--PartColl_Assembly3.png|600px|thumb|center|alt=domestication.|Step 3]]
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Latest revision as of 04:03, 18 December 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_K3893007, 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.

domestication.
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).

domestication.
Step 2

3. We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.

domestication.
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