Difference between revisions of "Part:BBa K4727009:Design"

 
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===Design Notes===
 
===Design Notes===
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The plasmid backbone has been deleted of five prohibited restriction sites, the region was substituted with the standard RFC[10] prefix and suffix. Between them has been placed the part <partinfo>BBa_J23119</partinfo>, a promoter that has been demonstrated to be working in <i> A. baumannii </i> and <i> K. pneumoniae </i>  
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The plasmid backbone has been deleted of five prohibited restriction sites, the region was substituted with the standard RFC[10] prefix and suffix. Between them has been placed the part <partinfo>BBa_J23119</partinfo>, a promoter that has been demonstrated to be working in <i> A. baumannii </i> and <i> K. pneumoniae </i>
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Given the results of our experiments and attempts in cloning a dCas expression cassette in <partinfo>BBa_K4727000</partinfo>, we decided to take a straightforward approach by directly changing the <i>E. coli</i> origin of replication and substituting it with a medium/low copy ORI. Since the plasmid from which we extract the dCas expression cassette carries a p15a ORI and we know it allows for dCas expression in <i>E. coli</i>, we decided that this sequence could be a possible solution.
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We decided then to exchange the plasmid pMB1 ORI with p15a. To operate this modification we opted for a Gibson assembly, for which we designed appropriate PCR primers pairs to amplify the plasmid backbone and the new ORI, flanked by homology sequences.
  
 
===Source===
 
===Source===
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===References===
 
===References===
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[1] Wang et al., 2019, Cell Chemical Biology 26, 1732–1742 December 19, 2019 ª 2019 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2019.09.003

Latest revision as of 10:47, 30 September 2023


Low copy backbone for K. pneumoniae and A. baumannii


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 4850
    Illegal suffix found at 4907
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4850
    Illegal NheI site found at 2155
    Illegal NheI site found at 4878
    Illegal NheI site found at 4901
    Illegal SpeI site found at 4908
    Illegal PstI site found at 4922
    Illegal NotI site found at 4856
    Illegal NotI site found at 4915
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4850
    Illegal BglII site found at 2348
    Illegal XhoI site found at 2961
    Illegal XhoI site found at 4966
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 4850
    Illegal suffix found at 4908
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 4850
    Plasmid lacks a suffix.
    Illegal XbaI site found at 4865
    Illegal SpeI site found at 4908
    Illegal PstI site found at 4922
    Illegal AgeI site found at 5462
    Illegal AgeI site found at 5785
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal SapI site found at 5194


Design Notes

The plasmid backbone has been deleted of five prohibited restriction sites, the region was substituted with the standard RFC[10] prefix and suffix. Between them has been placed the part BBa_J23119, a promoter that has been demonstrated to be working in A. baumannii and K. pneumoniae

Given the results of our experiments and attempts in cloning a dCas expression cassette in BBa_K4727000, we decided to take a straightforward approach by directly changing the E. coli origin of replication and substituting it with a medium/low copy ORI. Since the plasmid from which we extract the dCas expression cassette carries a p15a ORI and we know it allows for dCas expression in E. coli, we decided that this sequence could be a possible solution. We decided then to exchange the plasmid pMB1 ORI with p15a. To operate this modification we opted for a Gibson assembly, for which we designed appropriate PCR primers pairs to amplify the plasmid backbone and the new ORI, flanked by homology sequences.

Source

The plasmid backbone was acquired from AddGene and subsequently modified


References

[1] Wang et al., 2019, Cell Chemical Biology 26, 1732–1742 December 19, 2019 ª 2019 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2019.09.003