Part:BBa_K4294094

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].

[File: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