Difference between revisions of "Part:BBa K3893003"

 
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<partinfo>BBa_K3893003 short</partinfo>
 
<partinfo>BBa_K3893003 short</partinfo>
  
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Description: dsRNA designed for the target gene SGE1
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This sequence is part 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].
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dsRNA designed to silence the Velvet target gene via RNAi mechanism.
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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<!-- -->
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Once inside the pathogen, this dsRNA is further processed into RNA interference that can regulate the expression of any gene in the genome. SGE1 promotes the expression of SIX, which is required for the formation of conidia.  This has been a target gene to apply dsRNA against FOC R1 [1].
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==Assembly system==
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 +
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.
 +
 
 +
[[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]]
 +
 
 +
3. 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]]
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3893003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3893003 SequenceAndFeatures</partinfo>
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===References===
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[1] V. Gurdaswani, S. B. Ghag, and T. R. Ganapathi, “FocSge1 in Fusarium oxysporum f. sp. cubense race 1 is essential for full virulence,” BMC Microbiology, vol. 20, no. 1, p. 255, Aug. 2020, doi: 10.1186/s12866-020-01936-y.
  
  

Revision as of 03:53, 18 December 2021


dsRNA designed for the target gene SGE1

This sequence is part of the Modular Platform for dsRNA production in the proyect AgroBactory 593.

dsRNA designed to silence the Velvet target gene via RNAi mechanism.

Usage and Biology

Once inside the pathogen, this dsRNA is further processed into RNA interference that can regulate the expression of any gene in the genome. SGE1 promotes the expression of SIX, which is required for the formation of conidia. This has been a target gene to apply dsRNA against FOC R1 [1].

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 PstI site found at 438
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 438
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 438
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 438
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] V. Gurdaswani, S. B. Ghag, and T. R. Ganapathi, “FocSge1 in Fusarium oxysporum f. sp. cubense race 1 is essential for full virulence,” BMC Microbiology, vol. 20, no. 1, p. 255, Aug. 2020, doi: 10.1186/s12866-020-01936-y.