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===Usage and Biology===
 
===Usage and Biology===
  
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pTKEI-Dest: We utilized pTKEI-Dest as the acceptor vector of the construct we built to tackle the cross-talk issue among discrete senders’ subpopulations (more information about the cross-talk challenge in the Cross-talk section below). It includes a kanamycin (Kan) resistance gene and measures approximately 4.5kbp in length. It is a high-copy plasmid embodying the pBR322 origin of replication. Since this vector would have to be co-transformed with the plasmid “hosting” the final construct for senders, namely pTU2-A-RFP (p15A ori), it was specifically selected because of its origin of replication; p15A is classified as an incompatibility group B ori, therefore the second plasmid senders could carry would have to belong to either incompatibility group A or C. Taking into account the criteria agreed upon above alongside with this prerequisite, our search led us to the pTKEI-Dest vector, which, based on its ori, is categorized in incompatibility group A. As in pTU1-A-lacZ, pTKEI-Dest encompasses a lacZa gene, allowing for blue-white screening in agar plates containing x-gal and IPTG.
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pTKEI-Dest: We utilized pTKEI-Dest as the acceptor vector of the construct we built to tackle the cross-talk issue among discrete senders’ subpopulations (more information about the cross-talk challenge in the Cross-talk section below). It includes a kanamycin (Kan) resistance gene and measures approximately 4.5kbp in length. It is a high-copy plasmid embodying the pBR322 origin of replication. Since this vector would have to be co-transformed with the plasmid “hosting” the final construct for senders, namely pTU2-A-RFP (p15A ori), it was specifically selected because of its origin of replication; p15A is classified as an incompatibility group B ori, therefore the second plasmid senders could carry would have to belong to either incompatibility group A or C. Taking into account the criteria agreed upon above alongside with this prerequisite, our search led us to the pTKEI-Dest vector, which, based on its ori, is categorized in incompatibility group A. As in pTU1-A-lacZ, pTKEI-Dest encompasses a lacZa gene, allowing for blue-white screening in agar plates containing x-gal and IPTG [1,2].
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[[File:PTKEI-Dest Map.png]]
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''Plasmid Map''
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==References==
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[1] Addgene (plasmid #79784) https://www.addgene.org/79784/
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[2] Rapid construction of metabolite biosensors using domain-insertion profiling. Nadler DC, Morgan SA, Flamholz A, Kortright KE, Savage DF. Nat Commun. 2016 Jul 29;7:12266. doi: 10.1038/ncomms12266. 10.1038/ncomms12266 PubMed 27470466
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Latest revision as of 20:53, 13 October 2022


pTKEI-Dest

Usage and Biology

pTKEI-Dest: We utilized pTKEI-Dest as the acceptor vector of the construct we built to tackle the cross-talk issue among discrete senders’ subpopulations (more information about the cross-talk challenge in the Cross-talk section below). It includes a kanamycin (Kan) resistance gene and measures approximately 4.5kbp in length. It is a high-copy plasmid embodying the pBR322 origin of replication. Since this vector would have to be co-transformed with the plasmid “hosting” the final construct for senders, namely pTU2-A-RFP (p15A ori), it was specifically selected because of its origin of replication; p15A is classified as an incompatibility group B ori, therefore the second plasmid senders could carry would have to belong to either incompatibility group A or C. Taking into account the criteria agreed upon above alongside with this prerequisite, our search led us to the pTKEI-Dest vector, which, based on its ori, is categorized in incompatibility group A. As in pTU1-A-lacZ, pTKEI-Dest encompasses a lacZa gene, allowing for blue-white screening in agar plates containing x-gal and IPTG [1,2].

PTKEI-Dest Map.png

Plasmid Map

References

[1] Addgene (plasmid #79784) https://www.addgene.org/79784/

[2] Rapid construction of metabolite biosensors using domain-insertion profiling. Nadler DC, Morgan SA, Flamholz A, Kortright KE, Savage DF. Nat Commun. 2016 Jul 29;7:12266. doi: 10.1038/ncomms12266. 10.1038/ncomms12266 PubMed 27470466


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1911
    Illegal XbaI site found at 1884
    Illegal PstI site found at 1872
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1911
    Illegal PstI site found at 1872
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1911
    Illegal BamHI site found at 1890
    Illegal BamHI site found at 3720
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1911
    Illegal XbaI site found at 1884
    Illegal PstI site found at 1872
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1911
    Illegal XbaI site found at 1884
    Illegal PstI site found at 1872
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal SapI.rc site found at 178