Difference between revisions of "Part:BBa K3893012:Design"

 
 
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===Design Notes===
 
===Design Notes===
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Before constructing the modified plasmids and using them, we took into account the following:
 
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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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○ Previously carry out PCR overhang to the dsRNA sequence to add the restriction sites: BglII and KpnI.
 
  
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Once the above considerations have been met, the following assemblies are performed:
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In order to use, test and produce the dsRNA we design before, we created a plasmid backbone with an insert that is able to receive different sequences of dsRNA with a BglII and KpnI digestion.
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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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3) We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.
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 +
With this in mind, we face some shortcomings: first, it is a short fragment, with many sequence repeats, which makes it difficult for synthesis. Second, we want this insert to behave as a Biobrick compatible once it has the desired dsRNA sequence cloned inside.
 +
 +
It isn't compatible with RFC10 Assembly. Because the SpeI and BglII restriction sites are needed in the middle of the sequence for the assembly system.
  
  
 
===Source===
 
===Source===
  
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Assembly system
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Set of biobricks
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===References===
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Latest revision as of 04:17, 18 December 2021


Up part of cassette for dsRNA constitutive production 3


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 2525
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 749
    Illegal NheI site found at 1665
    Illegal NheI site found at 2470
    Illegal NheI site found at 3279
    Illegal SpeI site found at 2525
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2519
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 2525
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 2525
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 665
    Illegal BsaI.rc site found at 877
    Illegal BsaI.rc site found at 1581
    Illegal BsaI.rc site found at 2386
    Illegal BsaI.rc site found at 3195


Design Notes

In order to use, test and produce the dsRNA we design before, we created a plasmid backbone with an insert that is able to receive different sequences of dsRNA with a BglII and KpnI digestion.

With this in mind, we face some shortcomings: first, it is a short fragment, with many sequence repeats, which makes it difficult for synthesis. Second, we want this insert to behave as a Biobrick compatible once it has the desired dsRNA sequence cloned inside.

It isn't compatible with RFC10 Assembly. Because the SpeI and BglII restriction sites are needed in the middle of the sequence for the assembly system.


Source

Set of biobricks