Part:BBa_K4294092

pTU2-A-RFP (colE1 origin)

Plasmid Vector from the EcoFlex Modular MoClo kit.

Usage and Biology

We used pTU2-A-RFP (colE1 ori) as a level 2 (as indicated by the “2” in its name) - Golden Gate acceptor vector for assembling all different versions of the mNeonGreen/LuxR expression system in receivers: Open Loop (OL or OpLo), Positive Feedback (PF), Positive Feedback with a constitutive promoter (PFc), Positive Feedback with a Repressor (PF+R) and Positive Feedback with a Repressor and a constitutive promoter (PFc+R). This vector has a chloramphenicol (Cm) resistance gene and is a medium copy plasmid; it is approximately 3kbp in size. We selected a medium-copy plasmid for the expression of our system in receiver cells, as such a plasmid would allow the production of our visual output (mNeonGreen) while maintaining it at the levels receivers are actually activated by OC6 molecules; namely, without having a potentially high-copy vector counterbalance the different amount of AHL molecules receivers are stimulated by. This medium-copy backbone would not stress out cells carrying it either. Another reason we chose this particular vector was its marker; pTU2-A-RFP (colE1 ori) contains an RFP gene, enabling screening of colonies expressing recombinant plasmids without the need to use additional reagents (magenta colonies: transformed with the undesired parental plasmid, white colonies: transformed with a recombinant plasmid). [1] [2]

Plasmid Map

Reference

[1] Addgene plasmid # 74093 ; http://n2t.net/addgene:74093 ; RRID:Addgene_74093

[2] EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. Moore SJ, Lai HE, Kelwick RJ, Chee SM, Bell DJ, Polizzi KM, Freemont PS. ACS Synth Biol. 2016 May 2. 10.1021/acssynbio.6b00031 PubMed 27096716

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 107
  Plasmid lacks a suffix.
  Illegal XbaI site found at 140
  Illegal XbaI site found at 150
  Illegal PstI site found at 1259
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 107
  Illegal PstI site found at 1259
  Illegal NotI site found at 113
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 107
  Illegal XhoI site found at 2231
  Illegal XhoI site found at 3123
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 107
  Plasmid lacks a suffix.
  Illegal XbaI site found at 140
  Illegal XbaI site found at 150
  Illegal PstI site found at 1259
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 107
  Plasmid lacks a suffix.
  Illegal XbaI site found at 122
  Illegal XbaI site found at 140
  Illegal XbaI site found at 150
  Illegal PstI site found at 1259
  Illegal AgeI site found at 942
  Illegal AgeI site found at 1054
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 128
  Illegal BsaI.rc site found at 1253